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Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Le cancer est la principale cause de mortalité au Canada. Les taxanes (e.g. le paclitaxel et le docétaxel (DCTX)) constituent des remèdes efficaces contre une série de tumeurs solides telles que les cancers du sein, du poumon et de l’ovaire. Par ailleurs, des acides nucléiques (e.g. les oligonucléotides antisens (AON) ou les petits ARN interférents (siRNAs)), capables de supprimer sélectivement certains oncogènes impliqués dans la carcinogénèse, sont actuellement étudiés pour traiter une large gamme de cancers. Bien que l’activité des taxanes et des acides nucléiques soit bien établie sur des modèles humains et/ou animaux, plusieurs aspects physico-chimiques et cliniques restent encore à améliorer. Leur solubilité limitée (pour les taxanes), leur dégradation rapide dans le sang (pour les acides nucléiques), leur élimination précoce, leur absence de sélectivité et leur toxicité envers les tissus sains sont les principaux facteurs limitant leur efficacité. C’est pourquoi de nombreux efforts ont porté sur l’élaboration de systèmes de vectorisation ciblés à base de polymères, dans le but de surmonter les problèmes associés aux thérapies actuelles. Dans cette thèse, deux types de micelles polymères ont été développés pour la vectorisation de DCTX et d’acides nucléiques. D’une part, des micelles de poly(oxyde d’éthylène)-bloc-poly(oxyde de butylène/styrène) ont été étudiées pour la première fois pour solubiliser le DCTX et le protéger de l’hydrolyse. Ces polymères se sont révélés moins toxiques que le surfactant utilisé commercialement pour solubiliser le DCTX (i.e. polysorbate 80) et ont permis une libération prolongée du principe actif. D’autre part, deux systèmes différents de micelles polyioniques (PICM) ont été mis au point pour la vectorisation d’acides nucléiques. De nouveaux conjugués de poly(éthylène glycol) (PEG)-oligonucléotide ont été proposés pour la protection et la libération contrôlée d’AON. Lorsque ces conjugués ont été formulés avec des dendrimères de poly(amidoamine) (PAMAM), des complexes de taille homogène ont été obtenus. Ces PICM ont permis de prolonger la libération de l’AON et de le protéger efficacement contre la dégradation enzymatique. De plus, des polymères de poly(oxyde d’éthylène)-bloc-poly(méthacrylate de propyle-co-acide méthacrylique) ont été incorporés afin de conférer des propriétés acido-sensibles aux PICM. Dans ces micelles, formées de ce dernier polymère formulé avec le dendrimère PAMAM, des oligonucléotides (AON et siRNA) ciblant l’oncogène Bcl-2 ont été encapsulés. L’internalisation cellulaire fut assurée par un fragment d’anticorps monoclonal (Fab’) situé à l’extrémité de la couronne de PEG. Après l’internalisation cellulaire et la protonation des unités d’acide méthacrylique sous l’effet de l’acidification des endosomes, les micelles se sont affranchies de leur couronne. Elles ont ainsi exposé leur cœur composé d’acide nucléique et de dendrimère PAMAM, qui possède une charge positive et des propriétés endosomolytiques. En effet, ces PICM acido-sensibles ciblées ont permis d’augmenter la biodisponibilité des acides nucléiques vectorisés et se sont avérées plus efficaces pour silencer l’oncoprotéine Bcl-2 que les micelles non ciblées ou que le dendrimère de PAMAM commercial seul. Finalement, les nanovecteurs polymères présentés dans cette thèse se révèlent être des systèmes prometteurs pour la vectorisation des anticancéreux et des acides nucléiques. / Cancer is considered as the leading cause of premature death in Canada. Taxanes (e.g. paclitaxel and docetaxel (DCTX)) are effective against a range of solid tumors including breast, lung, and ovarian malignancies. In addition, nucleic acids (e.g. antisense oligonucleotides (AON) and short interfering RNA (siRNA)) which are capable of selectively suppressing oncogenes involved in carcinogenesis are currently being investigated for the treatment of a wide variety of cancers. Although the activity of taxanes and nucleic acid drugs is well-established in human and/or animal models, several physicochemical and clinical issues still need to be addressed. Low aqueous solubility (i.e. taxanes), rapid degradation in the blood (i.e. nucleic acids), fast clearance, non-selectivity and toxicity to normal tissues are limiting factors to their effectiveness. Hence, many efforts have been focused on developing targeted polymeric delivery systems to overcome the problems associated with the current therapies. In this thesis, two types of polymeric micelles have been developed for the delivery of DCTX and nucleic acids. On the one hand, poly(ethylene oxide)-block-poly(butylene oxide/styrene oxide) micelles were tested for the first time to solubilize and protect DCTX from hydrolytic degradation. The polymers showed less toxicity than the surfactant used commercially to dissolve DCTX (i.e. polysorbate 80) and released the drug in a sustained fashion. On the other hand, two different systems of polyion complex micelles (PICM) were developed for the sustained release and intracellular delivery of nucleic acids. Novel poly(ethylene glycol) (PEG)-oligonucleotide conjugates were assessed to protect AON against degradation and release them in a sustained manner. When these conjugates were mixed with poly(amidoamine) (PAMAM) dendrimers, monodisperse PICM were formed. These PICM further slowed down AON release and significantly protected it against enzymatic degradation. In addition, the incorporation of poly(ethylene oxide)-block-poly(propyl methacrylate-co-methacrylic acid) was exploited to impart pH-sensitivity to PAMAM-based PICM. This system was composed of the previous copolymer mixed with PAMAM dendrimer. Such PICM were loaded with AON or siRNA targeting the Bcl-2 oncogene. Micelles uptake by the cancer cells was mediated by a monoclonal antibody fragment (i.e. Fab') positioned at the extremity of the PEG corona. Upon cellular uptake and protonation of the methacrylic acid units in the acidic endosomal environment, the micelles lost their corona, thereby exposing their positively-charged endosomolytic PAMAM/nucleic acid core. The targeted, pH-sensitive PICM were found to increase the intracellular bioavailability of the entrapped nucleic acids and knock down the Bcl-2 oncoprotein more than either non-targeted micelles or commercial PAMAM dendrimers. The polymeric nanocarriers reported in this thesis appear to be promising vehicles for the delivery of anticancer drugs and nucleic acids.

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Medical devices, such as catheters and heart valves, are an important part of patient care. However, blood-contacting devices can activate the blood coagulation cascade to produce factor (f) Xa, the clotting enzyme that induces thrombin generation. By activating platelets and converting soluble fibrinogen to fibrin, thrombin leads to blood clot formation. Blood clots that form on medical devices create problems because they may foul the device and/or serve as a nidus for infection. In addition, clots can break off from the device, travel through the circulation and lodge in distant organs; a process known as embolization. This is particularly problematic with central venous catheters because clots that form on them can break off and lodge in pulmonary arteries, thereby producing a pulmonary embolism. Similarly, clots that form on heart valves can break off and lodge in cerebral arteries, thereby producing a stroke. Therefore, anticoagulants, blood thinning drugs, are frequently used to prevent clotting on medical devices. Conventional anticoagulants, such as heparin and warfarin, target multiple clotting factors. Heparin binds to antithrombin in plasma and accelerates the rate at which it inhibits fXa, thrombin and many other clotting enzymes. Warfarin, which is a vitamin K antagonist, attenuates thrombin generation by interfering with the synthesis of the vitamin K-dependent clotting factors, which include fX and prothrombin, the precursor of thrombin. In contrast to heparin and warfarin, more recent anticoagulants inhibit only a single clotting enzyme. For example, fondaparinux, a synthetic heparin fragment, only inhibits fXa and dabigatran, an oral thrombin inhibitor, only targets thrombin. Although effective for many indications, fondaparinux was less effective than heparin for preventing clotting on catheters in patients undergoing heart interventions and dabigatran was less effective than warfarin for preventing strokes in patients with mechanical heart valves. The failure of these new anticoagulants highlights the need for a better understanding into the drivers of clotting on medical devices. Therefore, the overall purpose of this thesis is to gain this understanding so that more rational approaches to its prevention can be identified. In the classical model of blood coagulation, clotting is triggered via two distinct pathways; the tissue factor (TF) pathway or extrinsic pathway and the contact pathway or intrinsic pathway; pathways which are initiated by fVIIa and fXIIa, respectively. The mechanism by which medical devices initiate clotting is uncertain. Platelet and complement activation and microparticle formation have been implicated, which would drive clotting via the TF pathway. Alternatively, medical devices can bind and activate fXII, thereby initiating the contact pathway. We hypothesized that medical devices trigger clotting via the contact pathway and induce the local generation of fXa and thrombin in concentrations that exceed the capacity of fondaparinux and dabigatran to inhibit them. To test this hypothesis, we used catheters as a prototypical medical device and we used a combination of in vitro and rabbit models. Several lines of evidence indicate that catheters initiate clotting via the contact pathway. First, catheter segments shortened the clotting time of human plasma, and this activity was attenuated in fXII- or fXI-deficient plasma, which are key components of the contact pathway, but not in fVII-deficient plasma, which is the critical component of the extrinsic pathway. Second, corn trypsin inhibitor (CTI), a potent and specific inhibitor of fXIIa, attenuates catheter thrombosis. Third, selective knockdown of fXII or fXI with antisense oligonucleotides attenuated catheter-induced thrombosis in rabbits, whereas knockdown of fVII had no effect. Therefore, these results revealed the importance of the contact pathway in device-associated thrombosis, and identified CTI or fXII or fXI knockdown as novel strategies for preventing this problem. Focusing on fXIIa as the root cause of medical device associated clotting, we coated catheters with CTI using a polyethylene glycol (PEG) spacer. In addition to unmodified catheters, other controls included catheters coated with albumin via a PEG spacer or catheters coated with PEG alone. Compared with unmodified catheters or with the other controls, CTI-coated catheters attenuated clotting in buffer or plasma systems and were resistant to occlusion in rabbits. These findings support the concept that catheter-induced clotting is driven via the contact pathway and identify CTI coating as a viable strategy for its prevention. We next set out to test the hypothesis that fondaparinux and dabigatran, which inhibit fXa and thrombin, respectively, are less effective than heparin, which inhibits multiple clotting enzymes. Fondaparinux and dabigatran were less effective than heparin at preventing catheter induced clotting and thrombin generation, respectively. Likewise, in a rabbit model of catheter thrombosis, fondaparinux was less effective than heparin and dabigatran was only effective when administered at doses that yielded plasma dabigatran levels similar to those found at peak in human given the drug; at trough levels, dabigatran was no better than placebo. Finally, we also showed synergy between heparin and either fondaparinux or dabigatran. Thus, when co-administered to rabbits in doses that on their own had no effect, the combination of fondaparinux or dabigatran plus heparin extended the time to catheter thrombosis. These findings support the hypothesis that when catheters trigger clotting via the contact pathway, fXa and thrombin are generated in concentrations that overwhelm the capacity of fondaparinux or dabigatran to inhibit them. Furthermore, the synergy between heparin and fondaparinux or dabigatran has clinical implications because it explains why supplemental heparin attenuated the risk of catheter thrombosis in patients treated with fondaparinux who underwent cardiac procedures and it identifies the potential role of supplemental heparin in dabigatran-treated patients who require such interventions. In summary, we have shown that catheters trigger clotting via the contact pathway and have identified CTI coating or fXII or fXI knockdown as viable strategies for prevention of this problem. In addition, for prevention of catheter thrombosis, we also have shown that heparin, which inhibits multiple coagulation enzymes, is more effective than fondaparinux or dabigatran, which only inhibit fXa or thrombin, respectively; findings consistent with the clinical observations. Moreover, the synergy that we observed between fondaparinux or dabigatran and heparin identifies supplemental heparin as strategy for preventing catheter thrombosis in patients receiving these drugs. Taken together, these studies provide insight into the mechanisms of catheter thrombosis and potential strategies for its prevention. / Thesis / Doctor of Philosophy (PhD)