Dissertations / Theses on the topic 'Anticoagulants'

Introduction. Devant les nombreux facteurs pouvant influencer le risque hémorragique despatients sous anticoagulant, le concept de médecine personnalisée pourrait avoir unimpact favorable dans la prise en charge globale de ces patients.Hypothèse et objectif. L’hypothèse de ce travail de thèse est que l’utilisation et l’analysede registre de « vraie vie » pourrait permettre de définir des « profils » hémorragique depatient permettant une prise en charge personnalisée des patients.Matériel et Méthode. Ce travail de thèse a utilisé deux registres de vraie vie, le registreRIETE (registre international multicentrique prospectif) et le registre RATED (registremonocentrique).Résultats. Nous avons montré l’importance du recueil biologique dans l’analyse desaccidents hémorragiques sous anticogulants avec une perte plus élevée de facteurs decoagulation, lors d’hémorragie gastro-intestinales par rapport aux intracraniennes sousAVK. A l’inverse, ce risque est diminué de moitié en cas de mutation du facteur V Leiden.Grâce au registre RIETE, nous nous sommes ensuite intéressés aux métrorragies sousanticoagulant (peu décrites dans la littérature) où seulement 0,17% des femmesprésentaient des saignements utérins majeurs. Nous avons par la suite montré que lespatients fragiles (CrCl ≤50 mL / min, un âge ≥75 ans ou un poids corporel ≤50 kg) ont unrisque deux fois plus élevé de saignement grave. Enfin, afin de montrer lacomplémentarité des registres de vraie vie et des données d’essais randomisés, nousnous sommes intéressés aux patients exclus de ces essais randomisés et avons montréégalement un risque hémorragique 4 fois plus élevé.Conclusion. Ce travail de thèse a permis de démontrer l’intérêt de travailler non pastoutes hémorragies confondues mais hémorragies par hémorragies, avec un intérêt deréalisation de registres prospectifs de vraie vie avec la mise en oeuvre de bio-banquepermettant une analyse plus personnalisée de la prise en charge des patients
Introduction. Given many risk factors that may influence the risk of hemorrhage underanticoagulant therapy, the concept of personalized medicine could have a favorableimpact in the overall management of these patients.Hypothesis and objective. The hypothesis of this thesis is that the use and analyze of "reallife" registries could allow to define hemorrhagic "profiles" allowing a personalizedmanagement of the patients.Material and method. This thesis work has used two real life registries, the RIETE registry(international, multicentric and prospective register) and RATED (monocentric register).Results. We showed the importance of biological database in the analysis of hemorrhagicevents under anticoagulants with a higher loss of coagulation factors in gastrointestinalbleeding compared with intracranial bleeding under AVK. Conversely, this bleeding risk istwo times lower in case of factor V Leiden mutation. Thanks to the RIETE registry, wewere interested in abnormal uterine bleeding under anticoagulant therapy (few studies inthe literature) with major uterine bleeding only for 0.17% of women. Then, we showed thatfragile patients (CrCl ≤50 mL / min, age ≥75 years or body weight ≤50 kg) have a 2-foldhigher risk for major bleeding. Finally, in order to show the complementarity between datafrom real life registries and randomized trial, we assessed patients normally excluded fromthese randomized trials and also showed a 4-fold higher bleeding risk in these excludedpatients.Conclusion. This thesis work allowed us to demonstrate the interest of working not only onoverall hemorrhages but on each type of hemorrhage separately, with a particular interestto create real life prospective registries with the implementation of bio-bank allowing amore personalized analysis of the intake of patients

Chemoenzymatically synthesized low molecular weight lignin polymers have been previously found to be potent inhibitors of a number of serine proteases via allosteric mechanisms targeting heparin binding sites. Herein, we describe the creation of synthetic sulfated β-O4 lignin (SbO4L) polymer, which is more homogenous compared to previous lignins with respect to its inter-monomeric linkage. SbO4L is a selective inhibitor of thrombin and plasmin. SbO4L was found to act via a unique mechanism targeting thrombin exosite 2 in a manner similar to platelet glycoprotein Ibα (GPIbα). Advanced hemostasis and thrombosis assays demonstrated that SbO4L acts via a dual mechanism: as an anticoagulant, by allosteric inhibition of thrombin catalysis; and as an antiplatelet agent, by competing with platelet GPIbα. These mechanisms are comparable in potency to low molecular weight heparins currently used in the market, indicating that targeting exosite 2 may yield clinically useful drugs in the future. Since the β-O4 type lignin was found to be selective for thrombin and plasmin, we hypothesized that other scaffolds from lignins could be potent inhibitors of other serine proteases. In particular, we screened a library of synthetic sulfated small molecules against factor XIa – an emerging target for prophylactic anticoagulation. Our search identified a sulfated benzofuran trimer (a mimic of β-5 type linkage found in lignins) as a potent inhibitor of factor XIa. Surprisingly, this inhibitor did not compete with heparin. A plausible binding site in the A3 domain of factor XIa was proposed by using molecular modeling techniques. The binding pose demonstrated good correlation with the structure activity data from in vitro studies. Further confirmation that the apple domains were required was proved by testing the trimer against recombinant catalytic domain. A 40-fold decrease in activity was observed. A temperature-dependant perrin plot demonstrated that factor XIa undergoes a large conformational change in the presence of the trimer, which is possibly converting the enzyme back into the zymogen-like shape. In general, the synthetic sulfated lignins can act as a useful foundation to develop anticoagulant, antiplatelet, and anti-inflammatory molecules in the future.

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Fundação para o Desenvolvimento Médico e Hospitalar (Famesp)
Estudo realizado, com base nos dados do ambulatório, para controle de anticoagulação nos pacientes portadores de próteses valvares cardíacas mecânicas do Serviço de Cirurgia Cardiovascular do Hospital das Clínicas da Faculdade de Medicina de Botucatu, no intervalo de dez anos, com os objetivos: avaliação da resposta à terapêutica profilática anticoagulante; quantificação das complicações tromboembólicas e hemorrágicas, com estratificação de sua gravidade; comparação entre tipos de anticoagulantes orais, doses e efeitos; influência da posição da prótese; presença de fibrilação atrial e tamanho do átrio esquerdo; e análise da estratégia de anticoagulação adotada. Foram incluídos, no estudo, 259 pacientes portadores de próteses mitrais (mitrais), aórticas (aórticos) e mitral e aórticas (mitro-aórticos). Foram analisadas 9714 consultas com valores do tempo de protrombina (em RNI) e dados dos registros hospitalares sobre complicações tromboembólicas e hemorrágicas com graus de gravidade. Os pacientes foram divididos em quatro grupos de acordo com o porcentual de consultas em que a RNI se encontrava dentro do intervalo desejado. Foram estudados dois anticoagulantes (Fenprocumona e Warfarina) e suas dosagens. Foi, também, avaliada a ocorrência de complicações tromboembólicas e hemorrágicas. Os resultados estão apresentados em: número de pacientes com complicações, estudo atuarial e freqüência linearizada de ocorrência de eventos. Os dados obtidos permitiram concluir que: a anticoagulação oral foi mais satisfatória nos aórticos do que nos mitrais e mitro-aórticos; os mitrais apresentaram ocorrência de eventos tromboembólicos semelhante a dos aórticos, porém com maior freqüência de hemorragias; os grupos que tiveram anticoagulação mais estável apresentaram menos complicações; poucas diferenças quanto à ocorrência de...
The study was held using outpatient data collected during 10 years for anticoagulation control of patients with mechanical prosthetic heart valves from the Cardiovascular Surgery Service at the Medical University Hospital in Botucatu city. The objectives were as follows: evaluation of prophylactic anticoagulation therapy, number of thromboembolic and hemorrhagic complications and their severity stratification, assessment of different oral anticoagulants, their effects and dosing, influence of prosthesis position, presence of atrial fibrillation and size of the left atrium, assessment of the anticoagulation strategy. Two hundred and fifty nine patients with mitral (M), aortic (A), mitral and aortic (M-A) prostheses were included in the study. Prothrombin time expressed in terms of international normalised ratio (INR) from 9714 consultations, medical data on thromboembolic and hemorrhagic complications as well as their severity were evaluated. Patients were allocated to four groups according to percentage of consultations which INR was within the target range. Two anticoagulants (Fenprocoumon and Warfarin), their dosing system, thromboembolic and hemorrhagic complications were also evaluated. Results were expressed as: number of patients with complications, actuarial study and linearized rate of events. Conclusions: oral anticoagulation was better in A than in M or M-A patients; M patients presented as many thromboembolic events as A patients although with higher hemorrhagic rate; groups presenting more steady anticoagulation showed fewer complications; there were few differences concerning complications among users of Fenprocoumon and Warfarin; most patients needed lower anticoagulant doses; patients with atrial fibrillation and/or enlarged left atrium presented as many thromboembolic complications as the other studied patients, although with higher... (Complete abstract click electronic access below)

Patients with atrial fibrillation (AF), a common arrhythmic disorder, are treated long-term with oral anticoagulants in order to prevent strokes. As warfarin treatment is associated with several problems, the direct oral anticoagulants (DOAC) dabigatran, rivaroxaban, apixaban and edoxaban have been introduced. However, real-world information regarding the utilisation of DOACs as well as their clinical effectiveness and safety is still scarce. Hence, the aim of this project was to increase the evidence from clinical practice regarding the use of DOACs in patients with AF in Scotland. Methods: This study has been designed as a retrospective cohort study (study period 2009 – 2015), using routinely collected administrative data. Three databases – the Prescribing Information System (PIS); Scottish Morbidity records (SMR); and National Records of Scotland (NRS) – covering prescriptions dispensed in primary care, hospital episodes and death records, respectively, have been linked using Community Health Index (CHI) numbers, a unique patient identifier in Scotland. Based on this data, three analyses have been conducted: a description of DOAC prescribing over time; an evaluation of patients’ adherence to DOAC treatment; and an analysis of the comparative clinical effectiveness and safety of DOACs. Results: In Scotland, the number of patients being treated with DOACs has steadily been increasing, and in 2015, the number of incident DOAC patients exceeded those of warfarin. During the study period, 14,811 AF patients with a mean age of 74.1 years [SD 11.3] initiated DOAC treatment. Adherence to treatment was good overall, with a median Medication Refill Adherence (MRA) of 102.3% [IQR 90.1% – 112.5%]; discontinuation rates were however variable, ranging from 24.9% (apixaban) to 63.3% (dabigatran). Persistence rates 12 months after treatment initiation were 61.8%, 78.6%, and 83.6% among patients initiating treatment with dabigatran, rivaroxaban, and apixaban, respectively. All DOACs were similarly effective in preventing strokes and systemic embolisms – nevertheless, the overall bleeding risk was higher with rivaroxaban as compared to apixaban [HR 1.52, 95% CI 1.21 – 1.91]. Conclusion: DOACs have swiftly been accepted into clinical practice, and adherence to treatment is generally good. As all DOACs are similarly effective, decisions for or against a specific drug should be made based on a wider risk assessment, with a focus on bleeding risks.

Les carboxymethyl dextranes benzylamide sulfonate/sulfate (cmdbs) sont des polysaccharides semi-synthetiques prepares a partir de dextrane par substitution statistique des fonctions hydroxyles par des fonctions carboxymethyles, benzylamides, sulfonates et sulfates. Huit cmdbs et un cmdsu presentant des taux de substitution eleves et des masses molaires differentes ont ete synthetises. Les cmdbs presentent un effet inhibiteur direct sur l'activite coagulante de la thrombine (t), sans inhibition de son activite amidolytique. Un effet catalytique des derives du dextran sur la reaction t/deuxieme cofacteur de l'heparine (hcii) mais pas sur la reaction t/antithrombine a ete mis en evidence. Les activites anticoagulante et catalytique de ces derives sont principalement liees a leur degre de sulfatation mais egalement a leur masse molaire. L'etude cinetique a permis de postuler que les cmdbs se lient rapidement a la thrombine, le complexe cmdbs-t constituant alors le complexe binaire responsable de l'augmentation de la vitesse de la reaction. Les cmdbs sont donc des polysaccharides sulfates anticoagulants presentant un mecanisme d'action double puisqu'ils interagissent preferentiellement avec la thrombine induisant a la fois une inhibition de l'interaction t-fibrinogene et une meilleure reactivite de l'enzyme vis a vis de l'hcii. Les fucanes sont des polysaccharides sulfates extraits d'algues brunes, doues d'activite anticoagulante et antithrombotique. Leur activite antithrombotique, observee dans des modeles de thrombose experimentale chez l'animal, suggere que ces extraits pourraient permettre le developpement de nouveaux medicaments antithrombotiques. Un effet proagregant direct, de trois extraits de fucanes, sur les plaquettes humaines a ete mis en evidence in vitro, aux concentrations testees. Par ailleurs, les anticorps anti pf4-heparine de cinq patients presentant une thrombopenie induite par l'heparine de type ii interagissent avec les trois extraits de fucanes

Purpose:This review summarizes the available evidence concerning direct oral anticoagulant (DOAC) use to treat venous thromboembolism (VTE) in patients with cancer as well as pertinent safety data on the use of DOACs in patients with both cancer and atrial fibrillation. Summary:The introduction of DOACs into clinical practice changed the way thrombotic complications are managed and prevented in diverse patient populations, including VTE and atrial fibrillation. Low-molecular-weight heparins have been the standard of care for treating VTE in cancer patients due to superiority over vitamin K antagonists in preventing recurrent VTE. Therefore, widespread DOAC use for VTE in patients with active cancer has not been adopted. Conclusion:Recent randomized clinical trials (SELECT-D, Hokusai VTE Cancer) have provided evidence that DOACs may have a role in treating VTE in cancer patients.

Trombotic diseases are a global health problem of multifactorial and multigenic etiology. They are characterized by the acute formation of trombus in veins and arteries. The oral anticoagulants are applied in order to create a partial deficiency on the active form of vitamin K, a co-factor in the changing of the factors of vitamin K-dependents coagulation, so that the risk of an abnormal coagulation can be reduced. The anti-vitamin K drugs or oral anticoagulants avoid the carboxylation of the II, VII, IX and X coagulation factors leading to the synthesis of the inactive factors. Several substances can change the action of oral anticoagulants, decreasing their action and increasing the thrombotic risk or increasing their activity and enlarging the blooding risk. The laboratory control of the oral anticoagulation is accomplished through the Time of Protrombine (TP) and of the Partial Thromboplastin Time (TTP) which are responsible for the success and safety of this therapeutic procedure. The present work has analyzed the parameters regarding the laboratory accompaniment of patients in use of oral anticoagulants as well as their interference on the medicine effect aiming to reach a better security for this kind of treatment. We can notice that most of the patients have made use of a quite rich diet in vitamin K producing variations in the values of their Normalized International Reasons (INRs) and consequently a larger difficulty in finding the right dose of the medicine for each patient. It also may be concluded that the anticoagulants response is influenced by some factors and those ones must be considered as, for example, the use of other medicines which affects the release or the absorption of the drug, laboratory technical troubles and the individual variability of each patient in relation to the drug.
As doenças trombóticas constituem um problema de saúde mundial de etiologia multifatorial e multigênica. São caracterizadas por formação aguda de trombos em veias e artérias. Os anticoagulantes orais são usados para criar uma deficiência parcial da forma ativa da vitamina K, um cofator na modificação dos fatores da coagulação vitamina K-dependentes e, assim, reduzir o risco de uma coagulação anormal. Os fármacos antivitamina K ou anticoagulantes orais impedem a carboxilação dos fatores II, VII, IX e X da coagulação, levando à síntese de fatores inativos. Uma série de substâncias pode alterar a ação dos anticoagulantes orais, reduzindo sua ação e aumentando o risco trombótico ou aumentando sua atividade e elevando o risco de sangramento. O controle laboratorial da anticoagulação oral é feito através do Tempo de Protrombina (TP) e do Tempo de Tromboplastina Parcial (TTP), os quais são responsáveis pelo sucesso e segurança deste procedimento terapêutico. Neste trabalho, foram analisados os parâmetros relativos ao acompanhamento ambulatorial de pacientes em uso de anticoagulantes orais e a interferência destes na atividade do fármaco em questão, buscando, dessa forma, uma melhor segurança para este tipo de tratamento. Observo-se que grande parte dos pacientes fazia uso de uma dieta bastante rica em vitamina K, o que acarretava variações nos valores de suas Razões Normalizadas Internacionais (INRs) e, com isso, uma dificuldade maior em encontrar a dose adequada do medicamento para cada paciente. Também se pode concluir que a resposta dos anticoagulantes é influenciada por alguns fatores e estes devem ser considerados como, por exemplo, o uso de outros medicamentos que afetem a liberação ou absorção do fármaco, problemas técnicos de laboratório e a variabilidade individual de cada paciente frente ao medicamento.

Orientador: Marcos Augusto de Moraes Silva
Resumo: Estudo realizado, com base nos dados do ambulatório, para controle de anticoagulação nos pacientes portadores de próteses valvares cardíacas mecânicas do Serviço de Cirurgia Cardiovascular do Hospital das Clínicas da Faculdade de Medicina de Botucatu, no intervalo de dez anos, com os objetivos: avaliação da resposta à terapêutica profilática anticoagulante; quantificação das complicações tromboembólicas e hemorrágicas, com estratificação de sua gravidade; comparação entre tipos de anticoagulantes orais, doses e efeitos; influência da posição da prótese; presença de fibrilação atrial e tamanho do átrio esquerdo; e análise da estratégia de anticoagulação adotada. Foram incluídos, no estudo, 259 pacientes portadores de próteses mitrais (mitrais), aórticas (aórticos) e mitral e aórticas (mitro-aórticos). Foram analisadas 9714 consultas com valores do tempo de protrombina (em RNI) e dados dos registros hospitalares sobre complicações tromboembólicas e hemorrágicas com graus de gravidade. Os pacientes foram divididos em quatro grupos de acordo com o porcentual de consultas em que a RNI se encontrava dentro do intervalo desejado. Foram estudados dois anticoagulantes (Fenprocumona e Warfarina) e suas dosagens. Foi, também, avaliada a ocorrência de complicações tromboembólicas e hemorrágicas. Os resultados estão apresentados em: número de pacientes com complicações, estudo atuarial e freqüência linearizada de ocorrência de eventos. Os dados obtidos permitiram concluir que: a anticoagulação oral foi mais satisfatória nos aórticos do que nos mitrais e mitro-aórticos; os mitrais apresentaram ocorrência de eventos tromboembólicos semelhante a dos aórticos, porém com maior freqüência de hemorragias; os grupos que tiveram anticoagulação mais estável apresentaram menos complicações; poucas diferenças quanto à ocorrência de... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The study was held using outpatient data collected during 10 years for anticoagulation control of patients with mechanical prosthetic heart valves from the Cardiovascular Surgery Service at the Medical University Hospital in Botucatu city. The objectives were as follows: evaluation of prophylactic anticoagulation therapy, number of thromboembolic and hemorrhagic complications and their severity stratification, assessment of different oral anticoagulants, their effects and dosing, influence of prosthesis position, presence of atrial fibrillation and size of the left atrium, assessment of the anticoagulation strategy. Two hundred and fifty nine patients with mitral (M), aortic (A), mitral and aortic (M-A) prostheses were included in the study. Prothrombin time expressed in terms of international normalised ratio (INR) from 9714 consultations, medical data on thromboembolic and hemorrhagic complications as well as their severity were evaluated. Patients were allocated to four groups according to percentage of consultations which INR was within the target range. Two anticoagulants (Fenprocoumon and Warfarin), their dosing system, thromboembolic and hemorrhagic complications were also evaluated. Results were expressed as: number of patients with complications, actuarial study and linearized rate of events. Conclusions: oral anticoagulation was better in A than in M or M-A patients; M patients presented as many thromboembolic events as A patients although with higher hemorrhagic rate; groups presenting more steady anticoagulation showed fewer complications; there were few differences concerning complications among users of Fenprocoumon and Warfarin; most patients needed lower anticoagulant doses; patients with atrial fibrillation and/or enlarged left atrium presented as many thromboembolic complications as the other studied patients, although with higher... (Complete abstract click electronic access below)
Doutor

La triple teràpia antitrombòtica (TT), consistent en un anticoagulant oral i la doble antiagregació plaquetària (DAPT), és el tractament recomanat en els pacients amb fibril·lació auricular (FA) sotmesos a un intervencionisme coronari percutani (ICP) amb implantació d’stent. Les recomanacions per a l’ús dels anticoagulants orals d’acció directa (ACODs) són similars a les dels pacients en tractament amb AVKs, atès que han demostrat ser, com a mínim, igual d’eficaços i segurs per la prevenció de l’ictus isquèmic i els esdeveniments tromboembòlics en la FA, i es recomana l’administració de la TT amb AVK o amb ACOD com a tractament inicial. No obstant això, en la pràctica clínica real no existeixen dades concloents sobre el perfil d’efectivitat i la seguretat dels ACODs associats a la DAPT en pacients amb FA sotmesos a ICP. L’objectiu d’aquesta tesi és avaluar l’efectivitat i la seguretat del tractament amb TT amb ACODs en comparació amb la TT amb AVKs en pacients amb FA no valvular sotmesos a ICP després de 12 mesos de seguiment. S’ha dissenyat un estudi observacional retrospectiu en dos hospitals de tercer nivell de l’àmbit espanyol i s’han inclòs tots els pacients adults que van rebre tractament amb TT amb ACODs o AVKs durant els anys 2013 - 2016. Es va partir d’un registre inicial de 5269 pacients sotmesos a ICP, en el qual es van identificar 187 pacients amb FA que van rebre TT: 85 pacients van rebre un ACOD i 102 van rebre un AVK. En la variable principal de seguretat, es va trobar que la taxa d’esdeveniments hemorràgics totals als 12 mesos va ser significativament menor en el grup de pacients tractats amb ACODs en comparació amb els pacients tractats amb AVKs (12,9% en ACODs vs 31,4% en AVKs; HR ajustada, 0,39; IC del 95%, 0,19 - 0,83, p=0,014). En la variable principal d’efectivitat, la incidència d’esdeveniments adversos cardíacs majors MACE (trombosis de l’stent, IAM, nova TVR, ictus o embolisme sistèmic i mort cardiovascular) va ser del 16,5% dels pacients tractats amb ACOD i el 22,5% dels pacients tractats amb AVK, sense trobar-se diferències estadísticament significatives entre grups (HR ajustada, 0,67; IC del 95%, 0,33-1,37, p= 0,273). El present estudi és el primer amb una població de vida real que ha comparat l'eficàcia i la seguretat de l'ús de la TT antitrombòtica amb ACODs o AVKs després de l’ICP amb implantació d’stent en pacients amb FA, mostrant taxes d’hemorràgies més baixes en els pacients tractats amb ACODs, i un perfil d’eficàcia comparable entre els dos tipus d’anticoagulants orals. Aquest treball pretén aportar llum en aquest camp proporcionant dades comparatives de món real entre l’anticoagulació amb ACODs i AVKs.
Triple antithrombotic therapy (TT) is recommended for patients with non-valvular atrial fibrillation (AF) undergoing percutaneous coronary intervention (PCI). However, there is a lack of comparative data in a real-world clinical setting between patients receiving TT with direct oral anticoagulants (DOAC) and patients receiving TT with Vitamin K antagonists (VKA). The aim of this thesis was to compare the safety and efficacy of TT with DOAC or VKA after PCI in patients with AF at 1-year of follow-up. We designed an observational retrospective study in two tertiary care hospitals during 2013-2016. A total number of 187 consecutive with an indication for anticoagulation due to AF from an initial registry of 5,269 patients undergoing PCI were identified: 85 were discharged on TT with NOAC and 102 were discharged on TT with VKA. The primary safety endpoint was the occurrence of total bleeding events at 12 months, which was found to be significantly lower in the DOAC group compared with the VKA group (12.9% in DOAC vs 31.4% in VKA, adjusted HR, 0.39; 95% CI, 0.19 – 0.83, P = 0.014). The primary efficacy endpoint was the rate of major adverse cardiovascular events (MACE) which occurred in 16.5% of patients treated with DOAC and 22.5% of patients treated with VKA, with no significant differences between groups (adjusted HR, 0.67; 95% CI, 0.33–1.37, P = 0.273). This is the first study in a real-world population comparing the efficacy and safety of the use of TT with either DOAC or AVK in patients with AF undergoing PCI. The results provide signal of lower bleeding rates and similar efficacy of DOACs as compared to VKA, shedding light into this field by providing real-world comparison data of DOAC vs VKA.

In recent years non-vitamin K dependent oral anticoagulants (NOACs) have started to replace warfarin for treatment and prevention of deep venous thrombosis (DVT), pulmonary embolism (PE) and stroke in patients with and without atrial fibrillation. There is a need for a simple and rapid method to detect the presence of these drugs in patient plasma. To meet these new demands, MediRox is developing a screening assay based on a novel prothrombin time (PT) method for rapid detection of NOACs in plasma. The assay is semi-quantitative and by dividing the International Normalised Index (INR) from a NOAC sensitive PT method with the INR from a NOAC insensitive PT method, NOAC containing samples be detected while plasma from normal donors and with warfarin are excluded.   The purpose of this project is to develop prototypes of assay quality controls for detection of NOACs in plasma.   The results show that the method used for the NOAC control prototypes is applicable and the PT ratio is comparable to patient samples for the low, medium and high concentrations of NOAC. The effect of lyophilisation indicates that the PT ratios for the NOAC control prototypes were nearly unaffected by the lyophilisation. The in-use stability at room temperature (20-25oC) for all NOAC control prototypes were at least 24 hours.   The methodology for production needs to be further optimised to increase the commutability to patient samples with very high concentrations of NOAC. The data indicates that the effect of lyophilisation is minimal and the stability of the NOAC control prototypes are satisfying, which is promising for future product development of NOAC controls.

The aim of this PhD was to elucidate the factors that influence anticoagulation response to the oral anticoagulants, warfarin and non-vitamin K antagonist oral anticoagulants (NOACs). Identification of the factors which influence clinical response to these agents could lead to improvements in patient management and treatment safety. In a cross-sectional study, the mean TTR over a 12 months period was significantly higher in hospital monitored patients (78%) (P=0.001) compared to those monitored in general practice (71%) or at their homes (68%). Domiciliary monitored patients had the least TTR among the three groups although they had the highest numbers of dose changes and INR monitoring events. In a further longitudinal study of patients on warfarin for up to 14 years, the TTR according to age was significantly lower in home monitored patients, with this group having a higher probability of having poor anticoagulation control (TTR ≤65%). In an ex-vivo study, rivaroxaban in vitamin K deficient older subjects produced a greater prolongation of both PT and modified PT, and a greater suppression in the rate and amount of thrombin generation compared to vitamin K replete younger subjects. Therefore, poor vitamin K intake could play a role in the reported incidences of bleeding associated with NOACs We had previously demonstrated that daily vitamin K supplementation causes an increase in warfarin daily dose requirement which varies between different patients and is related to VKORC1 genotype. Based upon these observations we set out to test our hypothesis that patients on warfarin therapy with the VKORC1(-1639)GG polymorphism could have poorer anticoagulation control than those with GA or AA genotypes as a result of variable dietary intake of vitamin K. However, the study results failed to confirm this hypothesis. Patients on warfarin therapy scheduled for an invasive surgery have to stop taking the drug for a fixed number of days to avoid peri-operative bleeding. However, there is variance in the rate by which INR falls after stopping warfarin. I found that patients with CYP2C9 variant alleles (CYP2C9*2*2 or CYP2C9*2*3) were >8 times (95% CI = 2.25–33.25) more likely to have INR of ≥ 1.5 before the planned day of surgery than those with wild-type genotype. In a further study, patient age and CYP2C9 *2*2 & *2*3 ii genotype were found to significantly influence the time required to reach an INR of 1.5 following warfarin cessation. VKORC1 genotype had no effect on the rate of INR decline.

Les anticoagulants anti-vitamines K (AVK) sont des inhibiteurs du mécanisme de régénération de la vitamine K. Celle-ci est impliquée dans l'activation post-traductionnelle par gamma-carboxylation de nombreuses protéines, et notamment de quatre protéines essentielles à la coagulation. L'apport alimentaire en vitamine K n'étant pas suffisant pour remplacer le cycle de régénération, un cycle efficace de régénération de la vitamine K est nécessaire. Les AVK bloquant ce cycle, la gammacarboxylation des facteurs de la coagulation n'est plus assurée, ainsi, la production de facteurs de la coagulation utilisables est fortement diminuée, ce qui affecte, par conséquent, le potentiel de la coagulation sanguine. Une inhibition importante du cycle de régénération peut entraîner la mort par hémorragies, c'est dans ce but que sont utilisés les AVK dans la lutte contre les rongeurs. Depuis une trentaine d'années, il a été remarqué que les rats femelles étaient moins sensibles aux AVK que les rats mâles. Plusieurs hypothèses pourraient expliquer cette différence : (i) une activité microsomale responsable de la régénération de la vitamine K plus efficace chez les femelles; (ii) une élimination plus rapide des AVK par les rats femelles; (iii) des concentrations basales et des demi-vies des facteurs de la coagulation vitamine K dépendants plus élevées chez les femelles. Le but principal de ce travail est de déterminer l'origine de la différence de sensibilité des rats femelles aux AVK.Les résultats mettent en évidence des différences significatives entre les mâles et les femelles concernant les niveaux basaux et les cinétiques d'évolution certains facteurs de la coagulation, pouvant expliquer la plus faible sensibilité des femelles aux AVK
Vitamin K antagonists (VKA) are inhibitors of the recycling mechanism of vitamin K. Vitamin K is involved in the post-translational activation by gamma-carboxylation of vitamin K dependant proteins, and in particular four proteins essential to coagulation. Food intake of vitamin K is insufficient to substitute the recycling mechanism, an efficient cycle is necessary. If VKA block the recycling mechanism, gamma-caboxylation of vitamin K dependant clotting factors becomes is not done, thus the production of the active form of these clotting factors is diminished. Consequently, the blood coagulability is affected. An important inhibition of the recycling mechanism can kill by haemorrhage, it’s with this aim that VKA are used in rodent management. Over the past three decades, a slight resistance to VKA has been observed in female rats beside male rats. Some hypothesis can explain this difference: (i) a more efficient activity of female microsomes which are responsible of the vitamin K regeneration; (ii) a faster elimination of VKA in female rats; (iii) basal concentrations and half-lives of vitamin K dependant clotting factors are higher in female rats. The main purpose of this work is to identify the origin of the resistance of female rats to VKA.Our results pinpoint significant differencies between females and males concerning the basal levels and the evolution kinetics of some vitamin K dependant clotting factors. This might explain the weaker sensibility of female rats to VKA

J'ai étudié deux voies de modulation de l'apoptose. J'ai d'abord participé à l'étude des propriétés anti-apoptotiques de la protéine Hsp27, sous forme de gros oligomères, inhibe l'activation des caspases par le cytochrome c. Sous forme de petits oligomères et par la protection de l'actine dans certains cas, par un mécanisme indéterminé dans d'autres, Hsp27 peut aussi inhiber la translocation mitochondriale de la protéine pro-apoptotique Bid et la sortie du cytochrome c. Dans une seconde partie, j'ai montré que des anticoagulants stimulent l'induction de l'apoptose par un anticorps agoniste anti-Fas. Ce phénomène est accru dans des cellules sur-exprimant les protéines Hsp27 ou Bc12. La compréhension des mécanismes moléculaires sous-jacents nécessite d'autres études. Ces anticoagulants, combinés à des anticorps agonistes de Fas ou des ligands de mort, seraient de bons candidats pour développer des thérapies contre des maladies auto-immunes ou des cancers.

L'héparine, polysaccharide sulfaté jouant un rôle anti-coagulant essentiel dans la cascade de la coagulation sanguine est actuellement utilisé dans le traitement des thromboses. Cependant, l'utilisation de l'héparine peut provoquer des effets secondaires nuisibles au patient. Dans le but de la remplacer, nous avons réalisé la synthèse de nouveaux calixarènes hydrosolubles, mimétiques des glycosaminoglycannes naturels. Ceux-ci, comportant 4, 6 ou 8 unités phénoliques, sont porteurs de groupements suflonates sur la couronne supérieure et d'un groupement fonctionnel de type acide, amine ou amide sur la couronne inférieure. Nous avons ensuite étudié les propriétés structurales de ces calixarènes par RMN et cristallographie aux Rayons X. Les propriétés de complexation de ces calixarènes à l'état liquide en présence d'acides aminés et de tampons biologiques et à l'état solide avec des ions divalents, des acides aminés et autres molécules organiques ont été analysées. Ces études entreprises par RMN et HPLC ont démontré que ces calixarènes interagissent fortement avec les acides aminés basiques via des interactions électrostatiques. Dans le but d'étudier ces molécules dans les milieux physiologiques en vue d'une utilisation anti-coagulante, nous avons étudié le comportement de ces calixarènes en présence de protéine et notamment l'albumine, protéine abondante dans le sang. Nous avons également étudié la toxicité cellulaire de ces calixarènes en présence de cellules rouges extraites de sang humain. Ces résultats ont montré que ces calixarènes ne présentent aucune activité hémolytique. Enfin, le pouvoir anti-coagulant de ces calixarènes a été mesuré en présence de protéines inhibitrices de la thrombine tels que Héparien co-facteur II (HCII) et Antithrombine III (ATIII). L'activité anti-coagulante de ces calixarènes a montré un mécanisme passant par HCII et non passant par ATIII

Le traitement AVK concerne plus d’1% de la population française. Il représente un problème de santé publique majeur en raison de son importante iatrogénie. Les causes peuvent être notamment reliées à la complexité du parcours de soins des patients, à leurs erreurs, méconnaissances et incompréhensions. L’éducation thérapeutique (ETP) permettrait de contribuer à rendre ce patient autogestionnaire de ses propres risques. Cette recherche qualitative vise à proposer un modèle d’ETP en en identifiant les compétences d’auto-soins et d’adaptation à la maladie et en en précisant le programme, les méthodes pédagogiques et d’évaluation. Le protocole de recherche comporte une revue de la littérature sur les connaissances des patients, des entretiens de type semi-directifs de patients traités par AVK et de soignants-éducateurs, et enfin des entretiens avec un groupe d’experts. Trente-six entretiens associés à une revue extensive de la littérature ont permis d’élaborer un référentiel de huit compétences. Vingt-et-un objectifs pédagogiques découlent de ces compétences. Les principales difficultés des patients concernaient la mise en lien des concepts constitutifs du paradigme du traitement par AVK. Les huit compétences du référentiel correspondent à la gestion intelligible et sans danger d’un traitement anticoagulant. Les différents types de soignants-éducateurs envisagés pour ce modèle d’ETP se retrouvent dans le parcours de soins habituel des patients. Notre modèle pédagogique se veut applicable à différents contextes de soins. Les propositions tentent de répondre à la problématique de l’intelligibilité du traitement AVK et de rendre accessible aux patients l’éducation thérapeutique
The VKA treatment concerns more than 1% of the french population. It represents a major public health problem beacause of its consirable drug related iatrogny

Lupus anticoagulants (LA) have been identified as antiphospholipid antibodies which can alter certain membrane-related endothelial activities, resulting in increased thrombogenesis. The effects of LA-containing patient plasmas on selected thrombosis-related endothelial functions were examined; 68% of 25 patient plasmas exhibited significant antiphospholipid antibody (APA) IgG and/or IgM directed against one of the four phospholipids tested in vitro; 44% exhibited anti-endothelial antibody. Eighty percent of plasmas with anti-endothelial IgM exhibited APA reactive with phosphatidyl inositol; antiphospholipid IgG did not correlate well with antiendothelium IgG. Patient plasmas that significantly stimulated EC PGI2 secretion uniformly exhibited anti-phosphatidyl serine IgM.Multiple mechanisms of induction of LA, and strong association of anti-PS and anti-EC antibody with thrombosis and related disorders were observed.Ball State UniversityMuncie, IN 47306

Le cytochrome P450 2C9 (CYP2C9) métabolise 20% des médicaments et est impliqué dans le métabolisme de molécules à index thérapeutique étroit : warfarine (antagoniste de la vitamine K, AVK), acénocoumarol (AVK). Ce CYP est génétiquement polymorphique. Deux allèles CYP2C9*2 et CYP2C9*3 affectent la posologie de la warfarine. Les patients ayant un de ces allèles (métaboliseurs lents, ML) ont des posologies d'AVK (warfarine, acénocoumarol) significativement plus faibles que les patients sans ces mutations. Il n'y a aucun test de phénotypage pour détecter les ML. Nous avons essayé de mettre au point un test de phénotypage avec le diclofénac puis l'acénocoumarol. Aucune de ces molécules ne convient pour causes pharmacocinétiques (diclofénac) ou de la grande variabilité interindividuelle de la réponse pharmacodynamique (VIRPD) à l'acénocoumarol. Mais nos travaux montrent que l'allèle CYP2C9*3 sous sa forme homozygote accroît la réponse PD et est responsable de surdosages aux anticoagulants oraux. Il est inutile de rechercher cet allèle chez les Asiatiques où il est extrêmement rare. Les allèles du CYP2C9 n'expliquent qu'une partie de la VIRPD aux AVK
Cytochrome P450 2C9 (CYP2C9) metabolises 20 % of drugs and is involved in the metabolism of molecules with narrow therapeutic index: warfarin (antagonist of the vitamin K, AVK), acenocoumarol (AVK). CYP2C9 is genetically polymorphique. Two alleles CYP2C9*2 and CYP2C9*3 affect doses of warfarin. Patients carrying one of these alleles (poor metabolisers, PM) have significantly lower doses of AVK (warfarin, acenocoumarol) than patients without these mutations. There is no phenotyping test to identify PM. We tried to establish a phenotyping test using diclofenac then acenocoumarol. None of these drugs agrees for pharmacokinetic reasons (diclofenac) or for the great interindividual variability of the acenocoumarol pharmacodynamic answer. But our works show that the CYP2C9*3 allele under its homozygous shape increases the acenocoumarol pharmacodynamic answer and is responsible for overdoses in oral anticoagulants. It is useless to research CYP2C9*3 in the Asian population because of its rarity. CYP2C9 alleles explains only a part of the interindividual variability of the pharmacodynamic answer to oral anticoagulants

The occurrence of thrombosis in several pathophysiological conditions creates a huge need for anticoagulation therapy. Thrombin and factor Xa have been prime targets for regulation of clotting through the direct and indirect mechanism of inhibition. This work investigates chemo-enzymatically prepared oligomers of 4-hydroxycinnamic acids (DHPs) as potential anticoagulants. Oligomers were prepared through peroxidase-catalyzed oxidative coupling of 4-hydroxycinnamic acids. The products resulting from this reaction are called CDs, FDs and SDs. Structurally, these sulfated DHPs are unique and do not resemble any of the anticoagulants known in the literature.DHP oligomers were found to increase clotting times at concentrations comparable to heparin. Studies in blood and plasma show that DHPs possess an anticoagulation profile similar to enoxaparin. To understand the mechanism of action of DHPs, we studied the inhibition of thrombin, FXa, FIXa, and FVIIa in the presence and absence of antithrombin. CDs and FDs display a preference for direct inhibition of thrombin and FXa, and exhibit a high level of specificity over FIXa and FVIIa. In the presence of AT, CDs and FDs displayed weaker inhibition of FXa and thrombin suggesting that binding to AT is a competitive side reaction. SDs exhibited potent inhibition of FXa and thrombin in the absence of antithrombin, but was inactive against FIXa and FVIIa representing the best selectivity among the DHPs. For SDs, inhibition of all the pro-coagulant enzymes favored the antithrombin dependent pathway. Binding studies were performed to determine how CDs directly inhibits thrombin. Competitive binding studies suggest that CDs interacts with exosite II and disrupts the catalytic triad of thrombin. These results indicate that the preferred mechanism of CDs action is exosite II mediated allosteric disruption of thrombin. CDs appears to be the first exosite II mediated DTI and this represents a novel mechanism of inhibitor function. The inhibition characteristics of DHPs are unique and radically different in structure from all the current clinically used anticoagulants. To the best of our knowledge this dual mechanism of anticoagulation and unique binding mode has not been described as yet in literature and represents a novel strategy that our laboratory has discovered.

Thrombin is the key protease that regulates hemostasis; the delicate balance between procoagulation and anticoagulation of blood. In clotting disorders, like deep vein thrombosis or pulmonary embolism, procoagulation is up-regulated, but propagation of clotting can be inhibited with drugs targeting the proteases involved, like thrombin. Such drugs however, have serious side effects (e.g., excessive bleeding) and some require monitoring during the course of treatment. The reason for these side effects is the mechanism by which the drugs’ act. The two major mechanisms are direct orthosteric and indirect allosteric inhibition, which will completely abolish the protease’s activity. Herein we sought an alternative mechanism called allosteric, partial inhibition, that has shown promise to truly regulate coagulation. Partial inhibition through allosteric mechanisms are well described for membrane-bound and oligomeric proteins. However proteases, specifically monomeric proteases (i.e., thrombin), have not shown this phenomenon until now. A small library of coumarin-based sulfated allosteric modulators (CSAMs) was synthesized to target a surface region called exosite 2; mainly composed of highly positively charged residues surrounded by hydrophobic patches. Studies revealed a non-competitive mechanism of binding with a range of IC50s between 0.2-58 µM combined with inhibitory efficacies (ΔY) between 22-73%; indicative of allosteric, partial inhibition. The KD was determined for the most potent compound (3g; IC50 = 0.2 µM, ΔY = 47%) at 0.15 µM. 3g was observed to bind at exosite 2 through unfractionated heparin competition and thrombin mutant studies. Additional computational studies were in agreement with the mutant and competition studies, showing the sulfate of 3g binding within a pocket containing R126 and R233. Fluorescence quenching and antithrombin inactivation rate studies described a conformational change to thrombin’s active site in the presence of 3g, supporting reduction of thrombin’s catalytic efficiency, without complete inhibition of thrombin’s proteolytic activities. Coupled enzyme assays and gel electrophoresis showed that in the presence of 3g, hydrolysis of fibrinogen (IC50 = 0.51 µM, ΔY = 94%) and protein C activation (IC50 = 1.7 µM, ΔY = 91%) is fully inhibited. Alternatively, FXIII activation was shown to be only partially inhibited by the presence of 3g, and FXI activation did not show any significant activation or inhibition. 3g was also shown to be active in human plasma and whole blood, but requiring much higher concentrations to induce an anticoagulant effect. Mice studies looking at the effects of 3g in vivo showed that even at high concentrations, showed no abnormal bleeding or any other irregularities. This work highlights a novel occurrence regarding thrombin’s allosteric functionality against multiple endogenous substrates. This library of compounds may be useful in the future development of allosteric inhibitors and probes that pose little to no risk of bleeding events by inducing partial inhibition.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
The therapeutic use of Varfarin, the most common oral anticoagulant it is indicated in many cases, including the atrial fibrillation, cardiac valvular prostheses and venous trombolic disease. Many discussions still exist related to the suspension or not before tooth extraction. People who are for itâs suspension agree that it may increase the risk of hemorrhage, however the ones who prefer to maintain itâs use refer the high risk of tromboembolism. Due to the controversy related to the cronic use of oral anticoagulant before tooth extraction and what to use to control bleeding after extraction, we decided to perform a one center randomized clinical trial study to compare the effectiveness of the hemostasis using soaked gauze with tranexamic acid at 4,8% and plain gauze and the use of collagen sponge (HemosponÂ), using it inside the tooth socket after extraction. The sample was made of 84 surgical procedures performed in 38 patients who were under anticoagulant treatment and who needed at least one tooth extraction. The trial was divided in three groups regarding the method used to reach hemostasis after tooth extraction. In group I we used compression with soaked gauze with tranexamic acid at 4,8%; in group II we used collagen sponge (HemosponÂ) inside the socket while in group III we compressed the socket with dry gauze for 8 minutes. There were two cases of post surgical bleeding, being one from group I and one from group II. The data collected was evaluated thru SPSS 1.5 (Statistic Package of Social Science) program. All the statistical analysis performed were considered significantly when p was less than 5%. We used the Qui square X2 Test, Fisher Exact Test e Analysis of Variance (ANOVA) to verify the variables of the data. There was no statistically significant difference between the groups, related to bleeding (p>0,05). The compression with dry gauze and suture, compression with soaked gauze with trenaxamic acid at 4.8% and suture and the use of collagen sponge (HemosponÂ) in the tooth socket hold with suture showed similar efficacy to the control of post extraction bleeding in patients who are under anticoagulant treatment.
A terapÃutica com varfarina, o anticoagulante oral mais utilizado, està indicada em mÃltiplas situaÃÃes, incluindo a fibrilaÃÃo atrial, prÃteses valvulares cardÃacas e o tromboembolismo venoso. DiscussÃes ainda existem sobre a indicaÃÃo ou nÃo da sua interrupÃÃo prÃvia a realizaÃÃo de exodontias. Aqueles que defendem a parada de sua administraÃÃo baseiam tal decisÃo no risco aumentado de hemorragias, enquanto os que acreditam na manutenÃÃo da terapia ressaltam o risco de tromboembolismo. Em virtude das controvÃrsias acerca da realizaÃÃo de exodontias em pacientes que fazem uso crÃnico de anticoagulantes orais, alÃm da dÃvida de que mÃtodo empregar no controle do sangramento pÃs-exodontia, decidimos realizar um estudo do tipo ensaio clÃnico, unicÃntrico, randomizado com o objetivo de comparar a efetividade hemostÃtica local da compressÃo com gaze embebida ou nÃo em Ãcido tranexÃmico à 4,8% com o emprego da esponja de colÃgeno (HEMOSPONÂ) no interior do alvÃolo pÃs-exodontia. A amostra foi constituÃda por 84 procedimentos cirÃrgicos realizados em 38 pacientes sob terapia anticoagulante que necessitavam de pelo menos uma extraÃÃo dentÃria. A amostra foi dividida em trÃs grupos a depender do mÃtodo hemostÃtico local empregado para o controle do sangramento apÃs a extraÃÃo dentÃria. No grupo I utilizou-se a compressÃo com gaze embebida em Ãcido tranexÃmico a 4,8%; no grupo II introduziu-se no interior do alvÃolo uma esponja de colÃgeno (HemosponÂ); enquanto no grupo III, a compressÃo com gaze seca por 8 minutos foi o mÃtodo empregado. Em dois casos foi observado sangramento pÃs-operatÃrio sendo um paciente do grupo I e outro do grupo II. Os dados coletados foram consolidados e avaliados por meio do programa SPSS 15.0 (Statistic Package of Social Science). Todas as anÃlises estatÃsticas efetuadas foram consideradas significativas quando valor de p foi menor que 5%. Utilizou-se os testes Qui-Quadrado (XÂ), Teste Exato de Fisher e AnÃlise de VariÃncia (ANOVA) para verificar as diferenÃas entre as variÃveis. NÃo houve diferenÃa estatisticamente significante entre os grupos com relaÃÃo à ocorrÃncia de hemorragias (p-valor>0,05). A compressÃo com gaze seca associado à sutura, a compressÃo com gaze embebida com Ãcido tranexÃmico a 4,8% associada a sutura e o emprego da esponja de fibrina (HemosponÂ) intra-alveolar associado a sutura mostraram eficÃcia semelhante no controle do sangramento pÃs-exodontia em pacientes sob terapia anticoagulante.

La fibril.lació auricular és l’arítmia cardiaca més freqüent, amb una elevada morbimortalitat i impacte econòmic. Augmentant-ne la importància epidemiològica, s’espera que la seva incidència s’incrementi de forma molt marcada com a conseqüència de l’envelliment de la població. En aquesta tesi doctoral, estudio la fisiopatologia i el tractament de la fibril.lació auricular a través d’un abordatge experimental i clínic. L’exercici físic de molt elevada intensitat és una de les causes emergents de fibril.lació auricular, especialment en individus joves i de mitjana edat. La fisiopatologia n’és desconeguda. En aquesta tesi, en un model exeperimental d’exercici físic en rata, es demostra mitjançant tècniques in vivo i in vitro que la hipertonia vagal característica dels atletes és un factor clau en el desenvolupament de la fibril.lació auricular de l’atleta. El substrat aritmogènic induit per l’exercici és reversible, especialment la hipertonia vagal. Per contra, el remodelat estructural (dilatació auricular i fibrosi) reverteixen de forma molt més lenta o incompleta. Els microRNA són seqüències curtes de RNA, recentment descrites, que participen en la regulació post-transcripcional de l’expressió génica. Els microRNA participen en gran quantitat de processos fisiològics i patològics, entre els quals el desenvolupament de fibrosi en diferents teixits. Tanmateix, es desconeix la seva participació en el desenvolupament del substrat de la fibril.lació auricular. En aquesta tesis, s’estudia la participació de miR-21 en el remodelat auricular post-infart de miocardi ventricular. S’usa un model en rata en el que objectivem una elevada inducibilitat de fibril.lació auricular. Es demostra que miR-21 participa en el desenvolupament de fibrosi miocàrdica auricular, i que la seva inhibició és capaç de prevenir-ne el desenvolupament i la inducibilitat de fibril.lació auricular. Es postul.la miR-21 com a possible diana terapéutica en el tractament de la fibril.lació auricular. El tractament anticoagulant constitueix la base de la prevenció del risc embòlic en la fibril.lació auricular. Ocasionalment, els pacients diagnosticats de fibril.lació auricular pateixen malaltia coronària com a comorbilitat, i requereixen tractament antiplaquetari amb aspirina i tienopiridines. El risc hemorràgic i trombòtic de la triple combinació d’anticoagulació, aspirina i tienopiridina roman desconeguda. En aquesta tesi estudiem aquest problema en pacients sotmesos a intervencionisme coronari i mostrem que es presenta en un de cada vuit pacients. En el seguiment, els pacients als que se’ls prescriu la triple combinació antitrombòtica presenten una incidència significativament menor d’esdeveniments trombòtics que els que no la van rebre, amb una major incidència d’hemorràgies no greus com a contrapartida.
Atrial fibrilliation is the most common cardiac arrhythmia, with a high morbidity, mortality and economic burden. An aging population in the upcoming years will likely increase its incidence and intensificate its epidemiological importante. This doctoral thesis approaches the pathology and therapy of atrial fibrillation through clinical and experimental studies. Very high intensity exercise is an emerging cause of atrial fibrillation in young and middle-aged individuals. However, its pathophysiology is unknown, yet. Here, a rat model of endurance, very high intensity exercise is used to study its mechanisms. By mean of in vivo and in vitro techniques, vagal enhancement is shown to be key factor in the development of exercise-induced atrial fibrillation. Moreover, exercise-induced arrhythmogenic atrial substrate is reversible, particularly concerning vagal enhancement. In contrast, structural remodeling (atrial fibrosis and dilatation) slowly or uncompletely reversed. MicroRNAs are recently discovered short RNA sequences that regulate post-transcriptional gene expression. MicroRNAs are involved in a large number of physiological and pathological processes, including fibrosis development. However, it is unknown whehter they participate in atrial fibrillation substrate. In this thesis, miR-21 involvement in post-left ventricle myocardial infarction atrial remodeling is explored. A left ventricle myocardial infarction rat model with a high burden of atrial fibrillation inducibility is used. We show that miR-21 is involved in atrial fibrosis pathology, and its inhibition may prevent its development and atrial fibrillation inducibility. Mir-21 is postulated as a potential therapeutic target in the treatment of atrial fibrillation. Anticoagulant therapy is on the basis of thrombo-embolic risk prevention in atrial fibrillation patients. Occasionally, patients diagnosed with atrial fibrillation are also diagnosed of ischemic cardiomyopathy and require antiplatelet therapy with aspirin and tienopiridines. The hemorrhagic risk of triple thrombotic combination of anticoagulation, aspirin and thienopiridines is unknown. In this thesis we study this problem in patients undergoing coronary intervention and show that this occurs in one out of eight patients in the cath lab. At follow-up patients who were prescribed triple combination antithrombotic had a significantly lower incidence of thrombotic events than those who did not receive it. A higher risk of non-serious bleeding balanced its benefits.

Les héparines regroupant les héparines standards (HNF), les héparines de bas poids moléculaire(HBPM), et le fondaparinux, sont des médicaments anticoagulants. Ils potentialisent l'antithrombine (AT) : un inhibiteur physiologique de la coagulation. Leur utilisation en thérapeutique est associée à un risque hémorragique majeur. Actuellement, le sulfate de protamine est le seul antidote disponible vis-à-vis des HNF. Il est partiellement efficace vis-à-vis des HBPM, et n'a aucun effet contre le fondaparinux, qui n'a pas d'antidote jusqu'à présent. C'est dans ce contexte que nous proposons des AT modifiées inactives, mais capables de se lier aux molécules d'héparines. Ces AT déplaceraient les molécules d'héparines de l'AT plasmatique, et neutraliseraient leur effet anticoagulant. Pour produire de telles AT, nous avons choisi une approche recombinante et une approche chimique. Dans la première approche, nous avons exprimé le variant AT-N135Q-Pro394. Ce variant possède une activité anti-Xa ou anti-IIa inférieure à 0,02% en présence de dérivés hépariniques, et une affinité à l'héparine 3 fois meilleure, comparée à l'AT plasmatique. En revanche, dans l'approche chimique, nous avons modifié l'AT plasmatique par la 2,3-butanedione (AT-BD), un réactif chimique de caractérisation des arginines. Contrairement au variant, cette AT-BD a une perte d'activité anticoagulante modérée, puis une affinité à l'héparine 20 fois meilleure, comparée à l'AT plasmatique. Malgré ces différences de propriétés biochimiques, ces 2 AT modifiées neutralisent d'une façon similaire les héparines in vitro et sur un modèle murin. Par ailleurs, à l'inverse du sulfate de protamine, nos antidotes n'ont pas d'activité anticoagulante propre sur un test de céphaline activée. Ainsi, ce travail de thèse a permis non seulement de proposer les premiers et les seuls antidotes spécifiques au fondaparinux décrits, mais aussi des antidotes alternatifs pour tous les anticoagulants hépariniques.

Les héparines regroupant les héparines standards (HNF), les héparines de bas poids moléculaire(HBPM), et le fondaparinux, sont des médicaments anticoagulants. Ils potentialisent l’antithrombine (AT) : un inhibiteur physiologique de la coagulation. Leur utilisation en thérapeutique est associée à un risque hémorragique majeur. Actuellement, le sulfate de protamine est le seul antidote disponible vis-à-vis des HNF. Il est partiellement efficace vis-à-vis des HBPM, et n’a aucun effet contre le fondaparinux, qui n’a pas d’antidote jusqu’à présent. C’est dans ce contexte que nous proposons des AT modifiées inactives, mais capables de se lier aux molécules d’héparines. Ces AT déplaceraient les molécules d’héparines de l’AT plasmatique, et neutraliseraient leur effet anticoagulant. Pour produire de telles AT, nous avons choisi une approche recombinante et une approche chimique. Dans la première approche, nous avons exprimé le variant AT-N135Q-Pro394. Ce variant possède une activité anti-Xa ou anti-IIa inférieure à 0,02% en présence de dérivés hépariniques, et une affinité à l’héparine 3 fois meilleure, comparée à l’AT plasmatique. En revanche, dans l’approche chimique, nous avons modifié l’AT plasmatique par la 2,3-butanedione (AT-BD), un réactif chimique de caractérisation des arginines. Contrairement au variant, cette AT-BD a une perte d’activité anticoagulante modérée, puis une affinité à l’héparine 20 fois meilleure, comparée à l’AT plasmatique. Malgré ces différences de propriétés biochimiques, ces 2 AT modifiées neutralisent d’une façon similaire les héparines in vitro et sur un modèle murin. Par ailleurs, à l’inverse du sulfate de protamine, nos antidotes n’ont pas d’activité anticoagulante propre sur un test de céphaline activée. Ainsi, ce travail de thèse a permis non seulement de proposer les premiers et les seuls antidotes spécifiques au fondaparinux décrits, mais aussi des antidotes alternatifs pour tous les anticoagulants hépariniques
Unfractionnated heparin (UFH), low molecular weight heparins (LMWH), and fondaparinux are used therapeutically as anticoagulants. They potentiate antithrombin (AT): a physiological inhibitor of coagulation. Their therapeutic use is associated with a major risk of bleeding. Currently, protamine sulfate is the only antidote available for UFH. It is partially effective for LMWH, and has no effect against fondaparinux, which has no antidote. So, we propose modified inactive AT, but able to bind heparin molecules as antidote of these heparins. These molecules would compete with plasmatic AT for binding to heparins, and neutralize their anticoagulant effect. To produce that AT, we realized a genetic approach and a chemical approach. In the first approach, we expressed the variant AT-N135Q-Pro394 that had an anti-Xa or anti-IIa activity below 0.02% in the presence of heparins, and heparin affinity three times higher, compared to the plasmatic AT. In the chemical approach, we modified the plasmatic AT by 2,3-butanedione (AT-BD), a chemical reagent for arginin’s characterization. The AT-BD had a moderate loss of anticoagulant activity, and a heparin affinity 20 times higher, compared to the plasmatic AT. Despite these differences in biochemical properties, these two modified AT neutralize similarly heparins in vitro and in a mouse model. Moreover, unlike protamine sulfate, our antidotes had not an intrinsic anticoagulant effect in activated partial thromboplastin test. Thus, this PhD-work offers the first and the only specific antidote described to fondaparinux, and it can be used too alternatively for all anticoagulant heparins

Les fucanes sont des polysaccharides sulfatés extraits d'algues brunes et doués d'activité anticoagulante. Les carboxymethyl dextrane benzylamide sulfonates (cmdbs), polysaccharides synthétiques également pourvus d'une activité anticoagulante sont préparés à partir de dextrane par substitution statistique de fonctions hydroxyles par des fonctions chimiques carboxymethyles, benzylamides, sulfonates et sulfates. L'activité anticoagulante des fucanes et des cmdbs dépend de leur composition chimique, notamment du taux de groupements sulfates, et de leur masse molaire. Ces composes catalysent préférentiellement la réaction d'inhibition de la thrombine par le second cofacteur de l'héparine, mais aussi par l'antithrombine. Le but de ce travail a été d'étudier les mécanismes d'action impliques dans l'activité anticoagulante de ces deux polysaccharides, comparés aux héparines. Des tests de génération de thrombine ont été réalisés en plasma humain en présence de fucane et de cmdbs. Une inhibition de la génération de thrombine est obtenue après activation de la phase contact en présence des deux polysaccharides, avec une phase de latence prolongée précédant la génération de thrombine, par rapport au témoin. La génération de thrombine obtenue après activation par le facteur tissulaire, est inhibée par le fucane et les cmdbs, avec un allongement de la phase de latence uniquement en présence de cmdbs. L'étude cinétique des réactions d'inhibition des facteurs ixa et xa par l'antithrombine a permis de mettre en évidence un effet catalytique faible de ces composes par rapport aux héparines. La détermination des constantes de dissociation des complexes du fucane ou du cmdbs avec chaque protéine (enzyme ou inhibiteur), associée à l'utilisation d'un modèle cinétique permet d'émettre l'hypothèse de la formation d'un complexe préalable du fucane avec l'inhibiteur, avant la formation du complexe stable enzyme-inhibiteur. Le cmdbs formerait un complexe préalable avec l'enzyme. L'activité antithrombotique du fucane a été mise en évidence dans un modèle de thrombose veineuse de type Wessler chez le lapin. Une dose vingt fois plus importante de fucane que d'héparine est nécessaire pour obtenir le même effet antithrombotique, mais l'effet du fucane est plus prolongé

Lupus anticoagulants (LA) are immunoglobulins (IgG, IgM, IgA or a mixture) that interfere with in vitro phospholipids (PL) dependent coagulation tests. Variable test results among LA positive individuals are due to the heterogeneous nature of the antibodies and differences in reagent/instrument systems. A laboratory’s ability to differentiate LA from other coagulation abnormalities and to accurately establish PL dependence is determined by the sensitivity and responsiveness of screening assays and specificity of confirmatory tests. The Third International Survey on Lupus Anticoagulants (ISLA-3) was organized to assess current protocols for LA testing and determine the sensitivity and specificity of confirmatory tests. A written survey and samples for evaluation were sent to 41 participants from five continents. A majority of laboratories performed 2 screening tests and all performed mixing studies if a screening test was abnormal. Confirmatory tests were done if mixing studies suggested the presence of an inhibitor. New confirmatory assays proved to be more specific than methods previously in place.Ball State UniversityMuncie, IN 47306
Center for Medical Education

A varfarina é um anticoagulante oral, antivitamina K, amplamente utilizado na prevenção de trombos intravasculares de diferentes etiologias. Exige um controle rigoroso e complexo em decorrência da sua interação com outros fármacos e alimentos, o que pode interferir na Qualidade de Vida Relacionada à Saúde (QVRS) dos usuários. Intervenções educativas têm sido utilizadas para diminuir o impacto dessa terapia na QVRS. O objetivo principal deste estudo foi comparar o efeito na QVRS de duas intervenções educativas em pacientes que iniciaram o uso de varfarina pela primeira vez durante a internação, aos três e seis meses após a alta. Como objetivos secundários, comparamos a presença de sintomas de ansiedade e depressão e a adequação do valor do International Normalized Ratio (INR) na faixa terapêutica indicada, entre os grupos. Este foi um estudo experimental com designação aleatória nos dois grupos. Os pacientes do grupo intervenção (GI) receberam o programa educativo com seguimento por telefone após a alta e os do grupo controle (GC) receberam o programa educativo sem o acompanhamento telefônico. O estudo foi aprovado por Comitê de Ética em Pesquisa e foi registrado na base ClinicalTrials.gov. A pesquisa foi conduzida no Hospital Estadual de Ribeirão Preto e no Hospital Estadual de Américo Brasiliense. Foram incluídos pacientes que iniciaram o uso da varfarina, pela primeira vez, maiores de 18 anos e com telefone para contato. O programa educativo foi norteado pela teoria de Bandura e composto por informações verbais e escritas sobre o tratamento, as quais foram apresentadas durante a internação. Os participantes do GI receberam cinco contatos telefônicos para o reforço dessas informações, após a alta hospitalar. Ambos os grupos tiveram dois encontros presenciais, aos três e aos seis meses, para avaliação das variáveis desfechos: QVRS (avaliada pela versão brasileira da Duke Anticoagulation Satisfaction Scale - DASS) e sintomas de ansiedade e de depressão (avaliados pelas subescalas da Hospital Anxiety and Depression Scale - HADS). Para comparar o DASS e as escalas do HADS, realizamos teste t de Student para amostras independentes. Para compararmos os grupos ao longo do tempo, em relação à QVRS, foi realizada Análise da Variância (ANOVA) para medidas repetidas, tendo como fatores o tempo (três e seis meses), o grupo (intervenção ou controle) e uma interação de tempo por grupo. O nível de significância adotado foi de 0,05. Os grupos eram similares quanto às caracterizações sociodemográfica e clínica. A maioria dos participantes eram casados, do sexo feminino e com média de idade de 55 anos (D.P=15). As indicações mais frequentes para o início da varfarina foram trombose venosa profunda e tromboembolismo pulmonar. Não encontramos diferenças estatisticamente significantes entre as médias de QVRS, ansiedade e depressão dos dois grupos, aos três e aos seis meses após a alta
Warfarin is an oral anticoagulant, antivitamin K, widely used in the prevention of intravascular thrombi of different etiologies. It requires rigorous and complex control as a result of its interaction with other drugs and foods, which may interfere with users\' Quality of Life Related to Health (HRQoL). Educational interventions have been used to lessen the impact of this therapy. The main objective was to compare the HRQoL, at three and six months after discharge, of two groups of patients who started using warfarin for the first time during hospitalization. Secondary objectives, were to compare the presence of anxiety and depression symptoms and the adequacy of the International Normalized Ratio (INR) value in the indicated therapeutic range, between the groups. It is an experimental study with random designation in two groups. The patients in the intervention group (IG) received the educational program with telephone follow-up after discharge and those in the control group (CG) received the educational program without telephone follow-up. The study was approved by the Research Ethics Committee and was enrolled in the ClinicalTrials.gov database. The research was conducted at the State Hospital of Ribeirão Preto and the Hospital Estadual de Américo Brasiliense and included patients who started using warfarin for the first time, over 18 years and with a telephone to contact. The educational program was guided by the Bandura Theory and was composed of verbal and written information about the treatment and approached during hospitalization. GI participants received five telephone contacts to reinforce this information after discharge. Both groups had two face-to-face meetings at three and six months for the evaluation of the outcome variables: HRQOL (assessed by the Brazilian version of the Duke Anticoagulation Satisfaction Scale - DASS) and symptoms of anxiety and depression (assessed by the subscales of Hospital Anxiety and Depression Scale - HADS). To compare the DASS and the HADS scales, we performed Student\'s t-test for independent samples. In order to compare the groups over time, in relation to HRQoL, we performed Variance Analysis (ANOVA) for repeated measures, taking as factors the time (three and six months), the group (intervention or control) and a time interaction per group. The level of significance was set at 0.05. The groups were similar in sociodemographic and clinical characterization. The majority of the participants were married, female and with an average age of 55 years (D.P = 15). The most frequent indications for the initiation of warfarin were deep venous thrombosis and pulmonary thromboembolism. We did not find statistically significant differences between the means of HRQoL, anxiety and depression of the two groups at three and six months after discharge

Os anticoagulantes orais são fármacos que agem aumentando o tempo de coagulação sanguínea, úteis na ocorrência de certas doenças que levam a formação de trombos intravasculares. Portanto, controles constantes dos níveis sanguíneos são necessários para um tratamento seguro. Algumas estratégias, como a intervenção educativa, vem demonstrando bons resultados. No presente estudo, o objetivo principal foi avaliar a qualidade de vida relacionada à saúde, adesão ao tratamento e autoeficácia de pacientes que iniciaram o uso de anticoagulantes orais segundo a participação em um programa educativo (Grupo Intervenção) ou o recebimento do cuidado de rotina (Grupo Controle). Como objetivos secundários, comparamos o estado de saúde percebido e a presença de sintomas de ansiedade e depressão entre os grupos. Trata-se de um estudo experimental com designação aleatória em dois grupos (Intervenção ou Controle). Há aprovação do Comitê de Ética em Pesquisa e registro na base ClinicalTrials.gov. Realizado no Hospital Estadual de Ribeirão Preto, foram incluídos pacientes que iniciaram o uso de anticoagulantes orais para tratamento clínico pela primeira vez, maiores de 18 anos e com avaliação cognitiva adequada. A estratificação e a aleatorização dos sujeitos foram obtidas por The Outpatient Bleeding Risk Index e por blocos, respectivamente. Envelopes coloridos foram usados na designação dos grupos. O programa educativo foi norteado pela Teoria de Bandura e composto por informações verbais e por escrito do tratamento dados durante a internação e contatos telefônicos para o reforço dessas informações na primeira e quarta semanas após a alta hospitalar do participante. Na coleta dos dados foram aplicados os instrumentos: caracterização sociodemográfica e clínica, Duke Anticoagulation Satisfaction Scale, Medidas de Adesão ao Tratamento, Hospital Anxiety and Depression Scale, Escala Visual Analógica. As análises foram feitas pelo IBM Statistical Package for Social Science (SPSS) version 21.0. Foram realizadas análises descritivas de frequência simples, de posição e de dispersão. Para a comparação das variáveis entre os grupos foi usado o teste T de Student não pareado. Para a comparação das frequências dos sintomas de ansiedade e depressão foi usada o teste Qui-quadrado ou o T de Student. Já a comparação das variáveis no início e no final do estudo em cada grupo foi obtida pelo teste T de Student pareado ou seu similar não paramétrico (teste de Wilcoxon). O Qui-quadrado foi aplicado para a comparação da ansiedade e depressão, antes e depois, em cada grupo. O nível de significância adotado foi de 5%. A comparação dos grupos mostrou características semelhantes, maioria de mulheres, casadas, idade próxima a 60 anos, Fibrilação Atrial ou Trombose Venosa Profunda como indicação para o início do anticoagulante oral. O Grupo Intervenção apresentou melhor qualidade de vida relacionada à saúde, maior adesão, maior autoeficácia diante do novo tratamento, o que possivelmente refletiu em menor ansiedade e depressão em relação ao Grupo Controle. Concluímos que a participação em um programa educacional proporcionou melhores desfechos em relação àqueles que receberam o cuidado de rotina.
Oral anticoagulants are drugs that act by increasing blood clotting time and are useful in the presence of certain diseases that lead to the formation of intravascular thrombi. Thus, constant controls of blood levels are necessary for a safe treatment. Some strategies such as educational intervention are showing good results. The main objective of the present study was to assess the quality of life related to health, adhesion to treatment and self-efficacy of patients who had started the use of oral anticoagulants according to the participation in an educational program (Intervention Group) or receiving routine care (Control Group). Secondary objectives were to compare the health status perceived and the presence of anxiety and depression symptoms between groups. This was an experimental study of random design conducted on two groups (Intervention and Control). The Research Ethics Committee approved the study, which was registered in the ClinicalTrials.gov database, and all subjects gave written informed consnet to participate. The study was conducted at the State Hospital of Ribeirão Preto and included patients who had started the use of oral anticoagulants for clinical treatment for the first time, who were older than 18 years and who had adequate cognitive function. The subjects were stratified and randomized using the Outpatient Bleeding Risk Index and by block, respectively. Colored envelopes were used for group allotment. The educational program was based on Bandura\'s theory and consisted of verbal and written information about the treatments applied during hospitalization and of telephone contacts for the reinfordcement of this information during the first and fourth weeks after hospital discharge of the patient. The following instruments were applied during data collection: sociodemographic and clinical characterization, Duke Anticoagulation Satisfaction Scale, Measurements of Adhesion to Treatment, Hospital Anxiety and Depression Scale, and Visual Analogue Scale. The analyses were carried out using the IBM Statistical Package for the Social Sciences (SPSS) version 21.0. Descriptive analyses of simple frequency, position and dispersal were performed. The unpaired Student t-test was used to compare the variables between groups, and the Chi-square or Student t-test was used to compare the frequency of anxiety and depression symptoms, while the paired Student t-test or the non-parametric Wilcoxon test was used to compare the variables at the beginning and at the end of the study in each group. The Chi-square test was applied to compare anxiety and depression before and after the study in each group. The levels of significance was set at 5%. Group comparison showed similar characteristics, a majority of women who were married and aged close to 60 years, and atrial fibrillation or deep venous thrombosis as the indication of the use of an oral anticoagulant. The Intervention Group showed better quality of life related to health, greater adhesion, and more self-efficacy when receiving the new treatment, a fact that possibly reflected lower anxiety and depression compared to the Control Group. We conclude that the participation in an educational program provided better outcomes compared to patients who received routine care.

L'objectiu principal d'aquesta Tesi doctoral ha estat l’obtenció d’una via alternativa a les existents per a l'obtenció d'un intermedi clau, 1, en la síntesi d'un fàrmac anticoagulant. La nova via havia de ser novedosa i patentable per tal de poder obtenir el producte industrialment. Així doncs inicialment es va fer una cerca bibliogràfica per tal de conèixer els precedents sintètics existents i a partir d'aquests es van proposar una sèrie d’objectius més concrets: una primera aproximació per tal de sintetitzar el ciclohexà central de 1, una altra basada en una reacció de iodolactamització per tal de tenir un dels nitrògens del sistema cis-diamino ja introduït i una tercera basada en una aziridina com a intermedi clau tenint en aquest cas tambè ja un dels nitrògens presents en la molècula. La primera aproximació es basava en una reacció Diels-Alder com a etapa clau per a formar el ciclohexà central de la molècula objectiu. Tot i arribar a un intermedi força avançat, no es va poder arribar a producte final i es va haver de descartar aquesta aproximació. La segona via que es va provar consistia en una reacció de iodolactamització com a etapa clau seguida de la formació del derivat de Boc. A partir d’aquest intermedi es van proposar vàries alternatives per a substituir l’àtom de iode i introduir el segon nitrogen a la molècula, ja fos directament amb amoníac per exemple, o a travès d’un equivalent sintètic del grup amina com podia ser l’azida o bè el grup ftalimida. Tambè es va provar de formar intermedis tipus urea amb isocianats i introduïr així el segon nitrogen de l’anell de sis baules intramolecularment. Malauradament cap d’elles va permetre arribar a obtenir el producte objectiu 1 així que es va passar a estudiar la darrera aproximació. La tercera via proposada passava per un intermedi clau aziridínic que es formava per reacció d’una olefina amb una sulfamida amb catàlisi de rodi. Desprès de provar la reacció principal a partir de diferents substrats, es va trobar un intermedi de tipus lactona inesperat però que va ser clau en aquesta aproximació. Seguint una sèrie de reaccions senzilles partint de la lactona es va arribar a obtenir 1. Així doncs es va assolir l’objectiu d’obtenir una via per tal d’obtenir 1. Malaurdament degut a una reivindicació d’una patent que protegeix l’últim intermedi de la síntesi i la transformació d’aquest a 1, no serà possible sintetitzar industrialment 1 seguint aquest nou procès obtingut. Tot i això, es va decidir seguir les reaccions de l’aproximació que havia permès obtenir 1, però variant el grup dimetilamida per altres grups alternatius. Es va pensar en formar èsters activats amb el 2,2,2-trifluoroetanol, el fenol i la N,N- dietilhidroxilamina. Aquesta estratègia permetria arribar a obtenir la molècula objectiu però sense infringir la patent ja que, en la darrera etapa, seria on s’introduiria el grup dimetilamida de manera que no es passaria per l’intermedi reivindicat. Malauradament, amb cap dels grups alternatius utilitzats es va aconseguir arribar a l’objectiu degut a la formació d’ intermedis tipus aziridina, els quals no es van poder transformar a la sulfamida bicíclica desitjada de cinc baules.
The main objective of this Thesis was to obtain a novel and alternative route to the existing key intermediate 1 in the synthesis of an anticoagulant drug. The new approach had to be innovative and patentable in order to produce the product industrially. First of all, we searched in the literature the existing synthetic precedents and from these we proposed a series of more specific objectives: a first approach to synthesize the central cyclohexane, another based on a iodolactamization reaction, having one of the nitrogens in the cis-diamino system already introduced, and a third approach based on aziridine as a key intermediate, in this case it has also already one of the nitrogens present in the molecule. The first approach was based on a Diels-Alder reaction as a key step to form the central cyclohexane target molecule. Despite reaching an advanced intermediate, this approach failed to reach the final product and had to discard it. The second route that was tested had a iodolactamization reaction as a key step and followed the synthesis of the Boc derivative of this product . From this intermediate we proposed several alternatives to replace the iodine atom and introduce the second nitrogen in the molecule, either directly with ammonia for example, or through a synthetic equivalent of the amino group for example with an azide or a phthalimide group. We also tried to form an urea intermediate with isocyanate and thus insert the second nitrogen in the six member ring intramolecularly. Unfortunately none of the methods allowed us to arrive at the desired product so we kept on studying the latest approach. The third proposed route passes through a key aziridine intermediate that was formed by the reaction of an olefin with a sulfamide using rhodium catalysts. After testing the main reaction from different substrates, we found a lactone intermediate which was key in this approach. Following a series of simple reactions starting from the lactone we get the objective molecule 1. The main objective was achieved, unfortunately, due to a claim of a patent that protects the last intermediate synthesis and transformation of this to 1, it will not be possible to synthesize 1 industrially following this new process. However, we decided to follow the reactions of the approach that had yielded 1, but varying the dimethylamide group to other alternative groups. We thought about forming activated esters with 2,2,2-trifluoroethanol, phenol and N,N- diethylhydroxylamine. This strategy would reach the target molecule obtained without infringing the patent because it was in the last stage, where it would introduce the dimethylamide group so the route would not pass through the intermediate claimed.

En el primer estudi confirmem que el tractament amb enoxaparina es capaç de disminuir la pressió portal en rates amb cirrosi establerta i hipertensió portal. A més, la millora en la pressió portal es va produir sense efectes en el flux sanguini portal, suggerint una disminució en la resistència vascular intrahepàtica. La falta d’efectes de l’administració aguda d’enoxaparina en la pressió portal, en els paràmetres hemodinàmics sistèmics, i en el contingut de superòxid o GMPc, suggereix que l’enoxaparina no té un efecte directe sobre el component dinàmic de la resistència vascular intrahepàtica. Per acabar de confirmar aquests resultats, es va examinar la resposta dependent d’endoteli al vasodilatador acetilcolina i no es van observar diferències entre les rates tractades amb enoxaparina i les rates tractades amb vehicle en cap dels dos models de cirrosi.. A més a més, tot i que en els tractaments de més llarga durada amb enoxaparina es van observar sobretot millores en les alteracions arquitecturals, es van observar també canvis en el to vascular. Es van disminuir els nivells d’estrès oxidatiu, de proteïnes nitrotirosinades i es va augmentar en el contingut de GMPc. A més a més, es va observar una disminució de la ratio p-Moesina/Moesina en les rates tractades amb enoxaparina, suggerint que el tractament amb enoxaparina pot modular el to vascular mitjançant la relaxació de les CHE, almenys a llarg termini. Tot i això, s’ha de tenir en compte que aquest canvis en el to vascular poden ser deguts a la millora de l’alteració estructural observada. És cert que el tractament amb enoxaparina ja es va descriure com a un bon tractament per a millorar la fibrosi hepàtica en models de dany hepàtic lleu. El nostre estudi amplia aquestes observacions a models amb cirrosi establerta. El tractament amb enoxaparina va ser iniciat en el moment on es va aturar l’administració del tòxic (tant CCl4 com TAA) com a model de regressió de malaltia avançada, o es va administrar alhora amb el tòxic, com a model de prevenció de la progressió de la cirrosi, en el model de TAA. Això ens va permetre analitzar els efectes del tractament amb enoxaparina en dos escenaris diferents. En el model de regressió amb CCl4, les rates que van rebre durant 3 setmanes el tractament van tenir més temps de regressió espontània de la fibrosi que les rates que van rebre només una setmana de tractament. Tot i això, tant les d’una setmana de tractament com les de 3 setmanes de tractament van tenir una disminució del 25% del contingut de col·lagen analitzat per tinció de Sirius Red. Això també es va observar en el model de cirrosi de TAA tant en regressió com en prevenció. Tot els resultats van cap a la mateixa direcció i suggereixen el paper de l’enoxaparina en disminució de la fibrosi hepàtica. A més, es va observar una disminució dels nivells d’activació de les CHE en les rates tractades amb enoxaparina, tant a nivell de teixit com en cèl·lula aïllada de rata tractada. A part de l’efecte de l’enoxaparina en la fibrosi hepàtica, es va observar la influència de del tractament en la prevenció d’esdeveniments trombòtics en la microcirculació hepàtica. De fet, s’ha suggerit que aquests podrien jugar un paper important en la progressió de la fibrosi hepàtica. Per explorar aquest efecte hem avaluat el contingut de fibrina en el parènquima hepàtic com a marcador de la formació de microtrombes. Els fetges de rates tractades amb enoxaparina van tenir una menor deposició de fibrina. Aquest fet ja es va observar en les rates tractades durant una setmana, però l’efecte va ser més gran i significatiu en les rates tractades durant 3 setmanes. Aquestes troballes confirmen la connexió que hi ha entre trombosi i fibrosi hepàtica i consolida el concepte de l’ús dels anticoagulants per a la prevenció/dissolució dels microtrombes per a millorar la fibrosi hepàtica. Per contra, no es van observar canvis a nivell d’inflamació. Per tant, encara que no podem descartar completament el potencial efecte del tractament amb enoxaparina en la reducció de la inflamació, no sembla que aquests sigui el principal mecanisme responsable de la millora les alteracions arquitecturals en la cirrosi hepàtica. És important remarcar que els efectes beneficiosos del tractament amb enoxaparina en la reducció de la pressió portal, resistència vascular intrahepàtica i fibrosi hepàtica no es van associar a efectes deleteris en els animals. No es van observar diferencies en els enzims hepàtics, ni es va observar sagnats durant el tractament o durant l’estudi hemodinàmic. En el segon estudi es va avaluar l’efecte de l’anticoagulant oral, Rivaroxaban, en la cirrosi hepàtica. Com ja es va veure en el primer estudi, la teràpia anticoagulant pot ser una bona estratègia terapèutica per al tractament de la hipertensió portal en la cirrosi. Tot i això, les heparines de baix pes molecular són dependents dels nivells sanguinis d’antitrombina, que de fet poden estar disminuïts en els pacients cirròtics. Es per això que vam estudiar els efectes de Rivaroxaban, que és un inhibidor directe del factor Xa (tant el Factor Xa lliure com el que es troba unit al complex de protrombinasa) i és independent dels nivells d’antitrombina. A més a més, Rivaroxaban té l’avantatge de ser biodisponible via oral i té una acció ràpida. Tot això, fa de rivaroxaban un anticoagulant més previsible i potencialment més eficient comparat amb altres heparines. En aquest estudi hem confirmat els efectes beneficiosos de l’ús d’anticoagulants per a millora la hipertensió portal en la cirrosi. Hem demostrat que l’administració oral de rivaroxaban disminueix els nivells d’estrès oxidatiu, millora la biodisponibilitat de NO, desactiva les CHE i millora la hipertensió portal en dos models de cirrosi en rata. La disminució de la pressió portal observada en les rates tractades amb rivaroxaban no es va associar a canvis en el flux sanguini portal en cap dels dos models, suggerint que el principal efecte ve de la disminució en la resistència vascular intrahepàtica. De fet, es va observar una disminució de la resistència vascular intrahepàtica en fetges de rates tractades amb rivaroxaban quan es va avaluar en un sistema de perfusió ex vivo amb el flux controlat. L’estudi dels mecanismes de la millora en la funció microcirculatòria hepàtica van mostrar que rivaroxaban disminuïa els nivells d’estrès oxidatiu, probablement pel menor reclutament de cèl·lules inflamatòries. També augmentava els nivells de GMPc, millorava la resposta a dosis creixents d’acetilcolina i disminuïa l’expressió del marcador d’activació de CES, el factor von Willebrand. Tot això indicava que el tractament amb rivaroxaban té un efecte important en la millora de la disfunció endotelial en el fetge cirròtic. D’altra banda també es va estudiar l’efecte de rivaroxaban sobre les CHE. Es va observar que el tractament amb rivaroxaban disminuïa els marcadors d’activació i proliferació de les CHE que correlacionen amb la seva capacitat de síntesi de matriu extracel·lular. Aquest efecte no es va associar a canvis importants en el contingut de fibra en el fetge. Degut als canvis observats en els nivells d’activació de les CHE, es temptador especular que un tractament més llarg amb rivaroxaban s’associaria a una disminució en els nivells de fibrosi hepàtica. Això està recolzat pels efectes beneficiosos observats a sobre les alteracions del parènquima: estudis ultraestructurals realitzats mitjançant microscopia electrònica van confirmar que les rates tractades amb rivaroxaban tenien una menor formació de membrana basal en els sinusoides hepàtics i que els hepatòcits tenen més projeccions, suggerint una millor estructura del sinusoid hepàtic. Finalment, es va confirmar una disminució del contingut de fibrina en les rates tractades amb rivaroxaban, suggerint una disminució de la microtrombosi hepàtica. Els efectes beneficiosos deguts al tractament amb rivaroxaban no es van associar a efectes deleteris en l’hemodinàmica sistèmica ni en un augment de sagnats durant el tractament. El tercer estudi aporta nova informació, fins ara no coneguda, sobre el potencial dany de l’estès oxidatiu mitocondrial en les cèl·lules hepàtiques durant la cirrosi i sobre l’ús dels antioxidants dirigits a mitocondri com a estratègia terapèutica per a la hipertensió portal i la cirrosi. Tal i com s’ha explicat anteriorment, les CHE són les principals cèl·lules implicades en la síntesi de matriu extracel·lular que contribueix en l’augment de la resistència vascular intrahepàtica en la cirrosi. Tenint en compte que l’augment de l’estrès oxidatiu en la cirrosi és un dels principals mecanismes que activen les CHE i que els mitocondris són un dels principals productors d’espècies reactives d’oxigen, aquest estudi es va centrar en avaluar els efectes de l’antioxidant dirigit a mitocondri, mitoquinona en l’estrès oxidatiu, fenotip de les CHE, la fibrosi hepàtica i la hipertensió portal en models pre-clínics de cirrosi. Els resultats del present estudi mostren que el tractament amb mitoquinona disminueix l’estrès oxidatiu hepàtic, desactiva les CHE i disminueix la fibrosi hepàtica i la hipertensió portal en les rates cirròtiques. Estudis anteriors van demostrar el rol de l’estrès oxidatiu en la fisiopatologia de la cirrosi i la hipertensió portal. En el present estudi demostrem que el tractament amb mitoquinona va aconseguir disminuir els nivells d’estrès oxidatiu, com altres teràpies utilitzades com ara el tempol o el resveratrol, però amb la diferència que la mitoquinona té l’avantatge de ser administrada oralment. A més a més, les altres teràpies antioxidants no poden dirigir-se a l’interior del mitocondri i, per tant, no poden contrarestar l’estrès oxidatiu mitocondrial que pot resultar en dany i disfunció mitocondrial i endegar una cascada de conseqüències deletèries per a la cèl·lula com ara la inflamació o la mort cel·lular. El nostre estudi demostra que part de l’estrès oxidatiu observat en els fetges cirròtics prové del mitocondris dels hepatòcits i de les CHE. A més, demostrem que la mitoquinona es capaç de disminuir l’estrès oxidatiu mitocondrial en els principals tipus cel·lulars hepàtics. Aquest augment de l’estrès oxidatiu mitocondrial en la cirrosi pot ser causat, almenys en part, per la disminució en l’expressió i l’activitat de la isoforma mitocondrial de la superòxid dismutasa, que s’ha vist disminuïda en la cirrosi. És important destacar que el tractament amb mitoquinona és capaç de disminuir l’activació de les CHE de rata tant in vitro com in vivo. A més, la desactivació de les CHE es va confirmar tant en CHE aïllades de biòpsies humanes, en la línia cel·lular humana de CHE, LX2, i en el model ex vivo per a l’estudi de mostres de fetge humanes, els talls de precisió de fetges humans (hPCLS). Aquests efectes es van evidenciar quan es va veure que la proliferació de les CHE estava disminuïda però no la seva viabilitat. Per confirmar la capacitat terapèutica de la mitoquinona, es va avaluar els seus efectes en dos models de cirrosi. Les observacions in vivo van demostrar que el tractament amb mitoquinona disminuïa la pressió portal, sense canvis en el flux sanguini portal, suggerint una disminució en la resistència vascular intrahepàtica. A més, els animals tractats amb mitoquinona van tenir una disminució del contingut de fibra hepàtica en ambdós models. Suggerint que la disminució de la fibrosi hepàtica és el principal mecanisme amb el que la mitoquinona disminueix la pressió portal, ja que no es van trobar efectes importants de la mitoquinona sobre el to vascular hepàtic almenys durant el temps de tractament estudiat, i que altres antioxidants sí que han demostrat una millora de la disfunció endotelial. A més, es sap que els nivells de les espècies reactives d’oxigen estan directament lligades a la inflamació. Per això vam analitzar l’expressió dels marcadors CD68 i CD163 com a marcadors d’inflamació. En aquest estudi vam observar una disminució del nombre de cèl·lules positives per CD68, suggerint un efecte de la mitoquinona en la reducció de la inflamació hepàtica. Aquests resultats només es van observar en el model de CCl4 i no en el de TAA, suggerint que la reducció de la inflamació pot contribuir a la millora de la fibrosi hepàtica però no és el principal mecanisme. Les diferències observades entre els dos models de cirrosi, pot ser degut a les diferències intrínseques dels dos models. De fet, s’ha descrit que el model induït per CCl4 acumula més estrès oxidatiu que el model per TAA, fent així el model per CCl4 més susceptible per a una teràpia antioxidant que no el model de TAA. Tot i això, cal destacar que tots els beneficis observats pel tractament amb mitoquinona no es van associar a efectes deleteris en els animals cirròtics en cap dels dos models.
The present doctoral thesis is focused in the development of new therapeutical strategies for the treatment of portal hypertension, one of the main complications of cirrhosis. Increase in portal pressure is directly related to the increased intrahepatic vascular resistance, mainly due to increased vascular tone and increased hepatic fibrosis. Therefore, the main objective of the present doctoral thesis was to study the use of the anticoagulants enoxaparin and rivaroxaban, and the mitochondrial-directed antioxidant mitoquinone and to evaluate their effects on intrahepatic vascular resistance and portal pressure in preclinical models of cirrhosis with portal hypertension. On the one hand, the results of the present doctoral thesis concluded that the anticoagulants, both the anti-thrombin like enoxaparin and the anti-factor Xa (thrombin-independent) rivaroxaban significantly improve portal pressure of animals with cirrhosis and portal hypertension mainly due to improved vascular tone and hepatic fibrosis. On the other hand, we observed that mitochondrial oxidative stress was increased in cirrhotic livers and hepatocytes and hepatic stellate cells were the main cells producing mitochondrial oxidative stress. Furthermore, we confirmed the beneficial effects of antioxidants on cirrhosis. The results concluded that the mitochondrial-directed antioxidant mitoquinone decreased significantly portal pressure and intrahepatic vascular resistance. Moreover, an important deactivation of the main cells producing collagen was observed both in rat and in human cells. These results suggest that anticoagulant and antioxidant treatment may be a potential therapeutical strategy for the treatment of portal hypertension in cirrhotic patients.
Esta tesis estudia el efecto de diferentes estrategias terapéuticas para el tratamiento de la hipertensión portal en models preclínicos de cirrosis. Se han estudiado los efectos de dos anticoagulantes (enoxaparina y rivaroxaban) y el efecto de un antioxidante dirigido a mitocondria (mitoquinona). En el primer estudio, se evaluó el efecto del anticoagulante enoxaparina en modelos preclínicos de cirrosis. Se observó que el tratamiento agudo con enoxaparina no afectó los parámetros sistémicos ni hemodinámicos evaluados. Tampoco modificó los niveles de GMPc o el contenido de estrés oxidativo hepático. El tratamiento a corto plazo (1 semana) disminuyó de manera significativa la presión portal en ratas cirróticas sin modificar el flujo. También disminuyó de manera significativa los niveles de estrés oxidativo y aumentó la biodisponibilidad de óxido nítrico (NO). Además, el tratamiento disminuyó un 26% la fibra hepática. Estos cambios se asociaron a una mejora del fenotipo de las células estrelladas hepáticas (CEH). También se observó una mejor en la microtrombosis hepática. El tratamiento a largo plazo (3 semanas) también disminuyó de manera significativa la presión portal en los dos modelos de cirrosis preclínica, sin cambios en los otros parámetros hemodinámicos. En este caso, también se observó una disminución del estrés oxidativo, pero sin mejora en la biodisponibilidad de NO. El tratamiento con enoxaparina redujo un 35% el área de fibrosis, cambios también asociados a una mejora del fenotipo de las CHE. También disminuyó el contenido de fibrina, mejorando así la microtrombosis hepática. Finalmente, también se observaron efectos beneficiosos en el modelo de prevención en ratas cirróticas por tioacetamida. La presión portal fue menor, en las ratas tratadas con enoxaparina, aunque los cambios no fueron tan marcados como en el modelo de regresión. También se redujo el área de fibrosis hepática, se mejoró el fenotipo de las CHE y hubo una menor microtrombosis hepática. En el segundo estudio, el anticoagulante oral anti factor Xa Rivaroxaban, disminuyó significativamente la presión portal en los dos modelos de cirrosis preclínica. La disminución de la presión portal no se asoció a cambios ni en el flujo sanguíneo portal ni en la presión arterial media, sugiriendo una disminución en la resistencia vascular intrahepática. Este hecho se confirmó ex vivo, donde se observó una disminución significativa de la resistencia vascular intrahepática del 35% en un sistema de perfusión con el flujo controlado. El tratamiento durante dos semanas disminuyó los niveles de estrés oxidativo, el número de células de Kupffer y aumentó la biodisponibilidad del NO. Las ratas tratadas con rivaroxaban tuvieron una mejor respuesta, aunque no significativa, al vasodilatador acetilcolina. Para confirmar el efecto beneficioso de rivaroxaban sobre el endotelio sinusoidal hepático, se evaluó el fenotipo de las células endoteliales sinusoidales y se observó una disminución en el marcador de activación, el factor von Willebrand, y una menor presencia de membrana basal. El tratamiento con rivaroxaban disminuyó el número y la activación de las CHE tanto a nivel de tejido como a nivel de célula aislada. No se observaron cambios en la apoptosis sugiriendo que el menor número de CHE vendría de una menor proliferación y no de un aumento en la muerte celular. Finalmente se observó que el tratamiento disminuía el contenido de fibrina sugiriendo una disminución de la microtrombosis hepática y se confirmó que los efectos de rivaroxaban sobre las CHE no era directos sino a través de la inhibición de la cascada de coagulación e inhibiendo la activación de los receptores PAR. En el tercer y último estudio se evaluó el efecto del tratamiento con un antioxidante dirigido a mitocondria. En un inicio se confirmó que en la cirrosis existe un aumento significativo de estrés oxidativo mitocondrial y que las principales células productores de éste estrés eran las CHE y los hepatocitos. El tratamiento con mitoquinona in vitro disminuyó significativamente el nivel de estrés oxidativo en todas las estirpes celulares hepáticas estudiadas. Además, disminuyó el nivel de activación de las CHE tanto de rata como humanas. Esto se confirmó en cortes ultrafinos de biopsias humanas y en líneas celulares. A nivel in vivo se observó que mitoquinona produjo un descenso significativo del nivel de estrés oxidativo hepático, tanto a nivel mitocondrial como total. A demás se disminuyó el contenido de proteínas nitrotirosinadas y la expresión génica de un factor inducido por el estrés oxidativo, Hif1a. Las ratas tratadas con mitoquinona tuvieron una disminución significativa de la presión portal y no se asoció a cambios ni en el flujo sanguíneo portal ni en la presión arterial media, sugiriendo una disminución en la resistencia vascular intrahepática. Mitoquinona también disminuyó el grado de fibrosis hepática y el nivel de activación de las CHE. Finalmente, se observó que el tratamiento con mitoquinona disminuyó significativamente el nivel de inflamación hepática. Para finalizar, esta tesis doctoral concluye que tanto el tratamiento con anticoagulantes, como el tratamiento con antioxidantes dirigidos a mitocondria pueden ser una potencial vía de tratamiento para la hipertensión portal en pacientes cirróticos.