Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

<p>Anticoagulant agents are commonly used drugs to reduce blood coagulation in acute and chronic clinical settings. Many of these drugs target the common pathway of coagulation because it is critical for thrombin generation and disruption of this portion of the pathway has profound effects on the hemostatic process. Currently available drugs for these indications struggle with balancing desired activity with immunogenicity and poor reversibility or irreversibility in the event of hemorrhage. While improvements are being made with the current drugs, new drugs with better therapeutic indices are needed for surgical intervention and chronic indications to prevent thrombosis from occurring.</p><p> A class of therapeutics known as aptamers may be able to meet the need for safer anticoagulant agents. Aptamer are short single-stranded RNA oligonucleotides that adopt specific secondary and tertiary structures based upon their sequence. They can be generated to both enzymes and cofactors because they derive their inhibitory activity by blocking protein-protein interactions, rather than active site inhibition. They inhibit their target proteins with a high level of specificity and bind with high affinity to their target. Additionally, they can be reversed using two different antidote approaches, specific oligonucleotide antidotes, or with cationic, “universal” antidotes. The reversal of their activity is both rapid and durable.</p><p> The ability of aptamers to be generated to cofactors has been conclusively proven by generating an aptamer targeting the common pathway coagulation cofactor, Factor V (FV). We developed two aptamers with anticoagulant ability that bind to both FV and FVa, the active cofactor. Both aptamers were truncated to smaller functional sizes and had specific point mutant aptamers developed for use as controls. The anticoagulant activity of both aptamer-mutant pairs was characterized using plasma-based clotting assays and whole blood assays. The mechanism of action resulting in anticoagulant activity was assessed for one aptamer. The aptamer was found to block FVa docking to membrane surfaces, a mechanism not previously observed in any of our other anticoagulant aptamers.</p><p> To explore development of aptamers as anticoagulant agents targeting the common pathway for surgical interventions, we fused two anticoagulant aptamers targeting Factor X and prothrombin into a single molecule. The bivalent aptamer was truncated to a minimal size while maintaining robust anticoagulant activity. Characterization of the bivalent aptamer in plasma-based clotting assays indicated we had generated a very robust anticoagulant therapeutic. Furthermore, we were able to simultaneously reverse the activity of both aptamers with a single oligonucleotide antidote. This rapid and complete reversal of anticoagulant activity is not available in the antithrombotic agents currently used in surgery.</p> / Dissertation

Diana.sobieraj@hhchealth.org / Objective To systematically review the literature and to quantitatively evaluate the efficacy and safety of extended pharmacologic treatment of venous thromboembolism (VTE) through network meta-analysis (NMA). Methods A systematic literature search (MEDLINE, Embase, Cochrane CENTRAL, through September 2014) and searching of reference lists of included studies and relevant reviews was conducted to identify randomized controlled trials of patients who completed initial anticoagulant treatment for VTE and then randomized for the extension study; compared extension of anticoagulant treatment to placebo or active control; and reported at least one outcome of interest (VTE or a composite of major bleeding or clinically relevant non-major bleeding). A random-effects Frequentist approach to NMA was used to calculate relative risks with 95% confidence intervals. Results Ten trials (n=11,079) were included. Risk of bias (assessed with the Cochrane tool) was low in most domains assessed across the included trials. Apixaban (2.5mg and 5mg), dabigatran, rivaroxaban, idraparinux and vitamin K antagonists (VKA) each significantly reduced the risk of VTE recurrence compared to placebo, ranging from a 73% reduction with idraparinux to 86% with VKAs. With exception of idraparinux, all active therapies significantly reduced VTE recurrence risk versus aspirin, ranging from a 73% reduction with either apixaban 2.5mg or rivaroxaban to 80% with VKAs. Apixaban and aspirin were the only therapies that did not increase composite bleeding risk significantly compared to placebo. All active therapies except aspirin increased risk of composite bleeding by 2 to 4-fold compared to apixaban 2.5mg, with no difference found between the two apixaban doses. Conclusion Extended treatment of VTE is a reasonable approach to provide continued protection from VTE recurrence although bleeding risk is variable across therapeutic options. Our results indicate that apixaban, dabigatran, rivaroxaban, idraparinux and VKAs all reduced VTE recurrence when compared to placebo. Apixaban appears to have a more favorable safety profile compared to other therapies. / Revisión por pares

<p>Thrombosis, or pathological blood clot formation, is intimately associated with cardiovascular disease and is the leading cause of morbidity and mortality in the western world. Antithrombotics are commonly prescribed as prophylactic medications or as rapid onset anticoagulants in acute care clinical settings. Although a number of antithrombotics are clinically available, their use is limited by immunogenicity, toxicity, and inability to be controlled with an antidote in the event of hemorrhage. Therefore, new antithrombotics that are effective, yet can be rapidly controlled are urgently needed. </p><p>Aptamers are oligonucleotides that form complex secondary and tertiary structures based on intramolecular base pairing and nucleic acid folding that allows them to bind to molecular targets with high affinity and specificity. Aptamers can be isolated that bind to proteins, such as clotting proteins, and modulate protein function. However, unlike most currently used antithrombotics, aptamers can be directly controlled with an antidote and therefore represent a safer class of therapeutic agents. </p><p>To generate a novel anticoagulant, we developed an aptamer-antidote pair against prothrombin. Prothrombin is a blood protein that plays an essential role in clot formation. I truncated, optimized, and studied the mechanism of an aptamer that can bind to prothrombin and inhibit prothrombin function, thereby severely impeding clot formation. Moreover, to increase the safety profile of this anticoagulant aptamer, I developed an antidote that can quickly reverse aptamer function and restore normal clotting. This aptamer and antidote pair is the first antidote reversible anticoagulant that targets prothrombin and may prove to be a valuable clinical anticoagulant.</p><p>A number of anticoagulants are in development, and a wide debate regarding the optimal protein target for anticoagulation is underway. We have previously generated anticoagulant aptamers to human coagulation factor VII, factor IX, factor X, and prothrombin. I compared the effects of these four anticoagulant aptamers to determine their impact on thrombin generation and clot formation. Each aptamer exerts its own unique effect on thrombin generation/clot formation, depending on the role that its protein target plays in coagulation. These studies provide valuable data regarding target validation and the anticoagulant effects of different therapeutic aptamers.</p><p>Robust anticoagulation is required during acute clinical surgical procedures to treat thrombosis. Currently used anticoagulants have several untoward side effects and most are not antidote controllable. I tested the effects of combining two anticoagulant aptamers to assess potential drug synergy. Several combinations of two anticoagulant aptamers were synergistic and severely impaired blood clot formation. One specific pair of aptamers that targeted factor X (FX) and prothrombin in combination was extremely potent and could keep blood fluid in an ex vivo model of extracorporeal circulation. Additionally, this pair of aptamers could be functionally modulated with two different types of antidotes. In conjunction with antidote reversal, this strategy of combining aptamer anticoagulants may prove useful in a variety of highly prothrombotic acute clinical settings. </p><p>Finally, to explore the potential of aptamers to regulate platelet function, I isolated and characterized an aptamer toward platelet glycoprotein VI. Glycoprotein VI is a platelet surface receptor that plays a key role in platelet activation and platelet plug formation. I isolated several aptamers that bind to glycoprotein VI, and show that the lead aptamer binds to platelets with high affinity and causes platelet activation and aggregation. This aptamer could potentially be further developed for topical administration to manage bleeding, or for biomarker detection of soluble glycoprotein VI in patient plasma.</p> / Dissertation

Thesis (M.S.M.) PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / BACKGROUND: Although novel anticoagulant drugs have proven safety and efficacy profiles from Phase III clinical trials, those patients with significant kidney disease were excluded. The lack of knowledge about incidence, severity and risk factors for severe renal dysfunction in patients requiring oral anticoagulation impedes development of strategies to mitigate risks of hemorrhage associated with renally-eliminated novel oral anticoagulants. METHODS: Patients taking warfarin for atrial fibrillation (AF) or venous thromboembolism (VTE) were consecutively enrolled from January 2007 to December 2010. Baseline kidney function was assessed and patients were followed to their first decline in Glomerular Filtration Rate (GFR) to < 30 ml/min estimated by the Cockcroft-Gault calculation. Independent risk factors for development of severe kidney dysfunction were assessed by multivariate analysis. RESULTS: Of 787 patients identified, 34 were excluded for baseline eGFR < 30 ml/min. The mean age of the cohort was 71 years. At baseline, 23% (n=174) had moderate renal impairment, or Stage 3 CKD (eGFR 30-59 ml/min), while 31% had mild disease. Overall, those with hypertension, congestive heart failure (CHF), diabetes mellitus (DM), and coronary artery disease (CAD) were 74%, 33%, 31%, 24%, and 9% of the cohort, respectively. A decline in eGFR to < 30 ml/min (the primary outcome) occurred in 91 patients, 25% of which happened within 5.3 months. Of those with baseline Stage 3 CKD, 37% experienced the primary outcome. In multiple logistic regression analysis, a baseline eGFR 30–59 ml/min conferred a greater than 14-fold increased risk in the development of eGFR < 30 ml/min (OR 14.5, 99% CI: 5.3 to 39.8, P<0.001) during the warfarin exposure period. CAD was associated with a greater than two-fold increased risk (OR 2.2, 95% CI 1.1 to 4.4, P=0.004). After adjusting for baseline kidney function, age was not an independent risk factor for a decline in eGFR to < 30 ml/min. CONCLUSIONS: Acute and chronic renal dysfunction is common among individuals on chronic warfarin therapy. Better understanding of the fluctuations in renal function would inform patient selection and monitoring strategies for optimal use of novel anticoagulants. / 2031-01-01

Venous thromboembolism (VTE) is a chronic illness that affects nearly 10 million people every year worldwide. Anticoagulant therapy with direct oral anticoagulants is the mainstay of treatment for patients with VTE, and should be continued for at least 3-6 months. Thereafter, a decision should be made to discontinue anticoagulation or continue it indefinitely. This decision is most challenging for patients with a first unprovoked VTE because of uncertainty in estimates for the long-term benefits (e.g., reduction in recurrent VTE) and harms (e.g., increase in major bleeding) of extended anticoagulation, and the trade-offs between them. The overarching aim of this doctoral thesis was to address these key evidence gaps that are pertinent to making decisions regarding the duration of anticoagulation for patients with a first unprovoked VTE. The first three studies of this thesis synthesized contemporary and reliable estimates for the long-term risks and consequences of recurrent VTE and major bleeding, with and without extended anticoagulation (parameters that can influence the clinical and cost-effectiveness of discontinuing versus continuing anticoagulation indefinitely). Broadly, these systematic reviews and meta-analyses found that: 1) the long-term risks and consequences of major bleeding during extended anticoagulation are considerable, particularly with vitamin K antagonists as well as in older patients, patients using antiplatelet therapy, and in patients with kidney disease, a history of bleeding, or anemia; and 2) the long-term risks of recurrent VTE during extended anticoagulation and major bleeding after discontinuing anticoagulation are reassuringly low but not negligible. The fourth study incorporated the synthesized evidence to compare the lifetime clinical benefits, harms, and costs of discontinuing versus continuing anticoagulation indefinitely. This decision analytic modelling study showed that indefinite anticoagulation is unlikely to either result in a net clinical benefit or be cost-effective in all (i.e., unselected) patients with a first unprovoked VTE. Findings from this thesis can serve to impact clinical practice and health policy by informing patient prognosis to guide shared decision-making regarding the duration of treatment for unprovoked VTE, and informing future research to ultimately identify which patients should receive anticoagulation indefinitely in order to maximize health benefits for the available healthcare resources.

Enoxaparin, a low-molecular-weight heparin, is commonly used as an anticoagulant in dogs, and is currently dosed at 0.8mg/kg every 6 hours. With an increase in individual enoxaparin doses, less frequent dosing may be possible, thereby reducing owner inconvenience and expense. The three phases of this study investigated the appropriate dose (Phase one- 0.8mg/kg, SQ once; Phase two- 2mg/kg, SQ once; Phase three- 1.3 mg/kg, SQ q8h for 7 total doses) and dosing interval needed for maximum effectiveness of enoxaparin. A Sonoclot® analyzer and factor Xa activity were used to assess level of anticoagulation in six healthy dogs. Anticoagulation was inconsistent at the 0.8mg/kg dose, while the 2mg/kg dose showed a high level of anticoagulation, and the 1.3mg/kg dose provided more reliable anticoagulation than the other dosages and dosing intervals. Small sample size and the use of same-breed healthy dogs potentially affected the strength of the results.

No description available.

Les pentasaccharides sont des inhibiteurs indirects du facteur Xa ayant des profils pharmacocinétiques très prédictibles. En raison de la liaison de forte affinité des pentasaccharides à l'antithrombine, cette pharmacocinétique peut être prédite mais aussi transférée à d'autres molécules qui leur sont liées de manière covalente. L'EP42675 combine dans une seule molécule, une antithrombine directe réversible peptidomimétique (analogue de l'α-NAPAP), et un pentasaccharide inhibiteur indirect du facteur Xa antithrombine dépendant (analogue du fondaparinux). L'EP217609 est le dérivé biotinylé de l'EP42675. Son action anticoagulante peut être neutralisée par l'avidine qui se lie avec une grande affinité et spécificité à la fraction biotine de l'EP217609. La première indication cible de l'EP217609 et de son antidote l'avidine est la chirurgie cardiaque nécessitant une circulation extracorporelle. La deuxième indication cible est le traitement des syndromes coronariens aigus nécessitant ou non une intervention coronarienne percutanée. Les études précliniques et cliniques de phase I et phase IIa résumées ici démontrent l'intérêt d'un tel concept de couplage avec un pentasaccharide : absence de dissociation entre les deux entités, faible variabilité intra et interindividuelle des paramètres pharmacocinétiques et pharmacodynamiques, et une neutralisation de l'activité anticoagulante de l'EP21609 quasi complète et sans effet rebond. / Pentasaccharides are indirect inhibitors of factor Xa with highly predictable pharmacokinetic profiles. Because of the high affinity binding of pentasaccharides to antithrombin, this pharmacokinetics can be predicted but also transferred to other molecules covalently bound to them. EP42675 combines in a single molecule, a reversible direct antithrombin (α-NAPAP peptidomimetic analog), and a pentasaccharide similar to fondaparinux with an indirect anti-factor Xa activity. EP217609 is the biotinylated derivative of EP42675 whose anticoagulant activity can be neutralized by avidin which binds with high affinity and specificity to the biotin moiety of EP217609.The first target indication of EP217609 and its antidote avidin is cardiac surgery requiring extracorporeal circulation. The second target indication is the treatment of acute coronary syndromes requiring or not a percutaneous coronary intervention.The preclinical and clinical Phase I and IIa studies summarized here demonstrate the value of such a coupling concept to the pentasaccharide: absence of dissociation between the two entities, low intra- and interindividual variability of the pharmacokinetic and pharmacodynamic parameters, and an almost complete neutralization of the EP217609 anticoagulant activity with no rebound effect.

Ce travail a consisté en la synthèse d’outils chimiques et au développement de leurs applications biologiques. Dans un premier temps, des sondes pour l’étude de la Topoisomérase IIA humaine ont été synthétisées. Ces sondes ont alors été testées sur lysat cellulaire pour la capture des protéines présentant une affinité pour ces médicaments. Dans un second temps, un nouveau lien clivable en conditions non dénaturantes pour des applications en protéomique chimique a été developpé. Ainsi, après optimisation de la structure, il a été intégré au sein d’une sonde d’affinité pour évaluer sa capacité de capture et libération de la PARP 1. Enfin, la réaction de click entre un azoture et un cyclooctyne a été appliquée à l’élimination d’une drogue circulante dans le sang.Après l’étude cinétique de la réaction, l’activité biologique et la pharmacocinétique des différents composés ont été évaluées pour optimiser la réaction de click in vivo. / The main goal of this work was to synthesize chemical tools and to developp their biological applications. In the first part, probes for the study of Topoisomerase II via chemical proteomic were synthesized. They were then used for pulldown experiments on cell lysats. In a second part, a new cleavable linker in non denaturing conditions was developped for chemical proteomic applications. After optimisation of the structure, it was incorporated in an affinity probe and tested for the pulldown of PARP 1. Finally, a click chemistry reaction, the SPAAC, was used to provok the elimination of a circulating drug. After the study of the kinetic of the reaction, the biological activity and the pharmacokinetic of the different compounds were evaluated to optimise the click reaction in vivo.