Current Medicinal Chemistry - Volume 11, Issue 17, 2004

Cardiovascular diseases, including thromboembolic diseases associated with the western life style are major causes of morbidity and mortality in our society. The major components of hemostatic system are thrombocytes, the coagulation proteins and the fibrinolytic system. Therapies for thromboembolic diseases are based on different approaches depending on the site in the vascular system that is at risk when the disease occurs. Antithrombotic therapy has progressed enormously over the last two decades. Enormous attention from academic as well as pharma companies and biotech industry has resulted in novel agents for the prevention and treatment of thromboembolic diseases (Reopro, Clopidogrel, low molecular weight heparins, Exanta, Actilyse). As evidenced by the observation that many companies have projects in different stages of discovery and development in their pipeline, current therapy still leaves enough room for the development of novel antiplatelet, anticoagulant, and thrombolytic agents. Attention on the improvement of new agents is focused on but not restricted to the development of more efficient therapies but special focus in this area is also given to the ease of use, no need for therapy monitoring, oral availability, duration of action and the absence of side effects such as bleeding. Recent developments in drug discovery technology, such as combinatorial chemistry, high throughput screening, molecular modeling, pharmacogenomic guided screening, gene inactivation strategies, new targets as well as enhanced development processes will most probably contribute significantly to the medical therapy in the future. A contemporary overview of the approaches that might lead to novel products and the available literature are reviewed in this issue. Approaches for the development of antiplatelet agents, anticoagulant therapies and profibrinolytic treatments are addressed by the authors. Our special thanks go to the contributors of this issue.

The hemostatic system comprises platelet aggregation, coagulation and fibrinolysis also termed primary, secondary and tertiary hemostasis. From the platelet transcriptome 6000 mRNA species and represent receptors, ion channels, signalling molecules, kinases, phosphatases, and structural, metabolic and regulatory proteins. This abundance of regulatory proteins points towards the importance of signal transduction in platelet function. First platelets adhere to collagen, this induces activation signals such as TXA2 that induces further Ca2+ increase. Consecutively, fibrinogen binds to the integrin αIIbβ3 resulting in aggregation.This self-amplifying process is controlled by signals, from endothelial cells, to restrict the platelet plug to the site of vessel injury. Secondary hemostasis (coagulation) consists of an extrinsic and intrinsic pathway. Thrombin is generated via Factor Xa resulting from the extrinsic tenase reaction that is turned of by tissue factor pathway inhibitor. While thrombin generation is maintained via positive feedback mechanisms activating factors V, VIII and XI. Excess thrombin is inhibited by antithrombin or by autodownregulation via activation of protein C. Since minor injuries are common, platelets and plasma clotting factors constantly produce clots to stop bleeding. If clots remained after the tissue healing, the vascular bed would become obstructed with clots therefore this is regulated by fibrinolysis, tertiary hemostasis. Tissue-type plasminogen activator synthesised by the endothelium, converts plasminogen to plasmin, the clot lysis enzyme. Plasmin clears the blood vessels by degrading fibrin. Fibrinolysis is controlled by plasminogen activators inhibitor (PAI-1), α2-antiplasmin and α2-macroglobulin, and thrombin-activatable fibrinolysis inhibitor (TAFI).

Cardiovascular diseases are one of the major causes of mortality in the western world. As platelet dependent thrombosis is of central importance in their pathophysiology, several successful strategies, targeting a specific platelet function or interaction, have been developed to prevent or treat these disorders. However, as the current antiplatelet strategies are limited in efficacy and safety, and often influence normal haemostatic functions, new compounds are being developed with improved characteristics. This review deals with the development of novel antiplatelet compounds for which evidence is available on their antithrombotic action in vivo. In a first part, these compounds, their targets and their potential applicability are discussed. The second part of this review focuses on BT tests and bleeding models and their usefulness for determination and / or prediction of the safety of novel antiplatelet compounds.

Factor VIIa (FVIIa) is a key serine protease involved in the initiation of the coagulation cascade. It is a glycosylated disulfide-linked heterodimer comprised of an amino-terminal ?-carboxyglutamic acid-rich (Gla) domain and two epidermal growth factor (EGF)-like domains in the light chain, and a chymotrypsin-like serine protease domain in the heavy chain. FVIIa requires tissue factor (TF), a membrane bound protein, as an essential cofactor for maximal activity towards its biological substrates Factor X, Factor IX and Factor VII (FVII). Inhibition of TF•FVIIa activity may prevent the formation of fibrin clots and thus be useful in the management of thrombotic disease. The development of TF•FVIIa inhibitors to validate this target has been of great interest. A wide array of strategic approaches to inhibiting the biochemical and biological functions of the TF•FVIIa complex has been pursued. This has been greatly aided from our understanding of the structures for TF, FVII, FVIIa, and the TF•FVIIa complex. These approaches have resulted in inhibitors directed specifically towards either FVIIa or TF. Antagonists include active site inhibited FVIIa, TF mutants, anti-TF antibodies, anti-FVII / FVIIa antibodies, naturally-occurring protein inhibitors, peptide exosite inhibitors, and protein and small molecule active site inhibitors. These antagonists can inhibit catalysis directly at the active site as well as impair function by binding to exosites that may interfere with substrate, membrane, or cofactor binding. The rationale of TF•FVIIa as a target and the development, characteristics and biological uses of TF•FVIIa inhibitors are discussed.

Current treatments for preventing thrombotic diseases are associated with a significant risk of bleeding. Improved anticoagulant agents are therefore still required. The specificity and pharmacokinetics properties of monoclonal antibodies to coagulation factors allow novel anticoagulation approaches. Treatment with human antibodies or humanised mouse monoclonal antibodies should avoid unacceptable side effects due to immune response to the drug. Such antibodies were developed against three coagulation factor: Tissue factor (TF), Factor IX (FIX) and Factor VIII (FVIII). A fully humanised antibody was successfully derived from a mouse monoclonal antibodies to TF. In vivo studies with monoclonal antibodies to TF demonstrated efficient antithrombotic activity. Anti-TF antibodies may also prove useful in cardiovascular disorders and cancer, given the role of TF in these diseases. Mouse and human monoclonal antibodies to FIX were also efficient to prevent thrombosis in animal models of venous and arterial thrombosis and in stroke. A humanised anti-FIX antibody was tested in phase I study in healthy volunteers. The pharmacokinetics of the antibody were determined by the rapid formation of stable complexes with newly synthesised FIX. Human anti-FVIII antibodies inhibiting only partially FVIII activity were recently described. Investigations in mice have established that treatment with such anti-FVIII antibodies is efficient to prevent deep vein thrombosis. Given the low concentration of FVIII in plasma and the long half-life of antibody, treatment with anti-FVIII antibody could be very convenient, allowing one administration every month. Altogether, monoclonal antibodies to coagulation factor appear as promising novel antithrombotic drugs.

The trypsin-like serine protease thrombin is a multifunctional key enzyme at the final step of the coagulation cascade and is involved in the regulation of hemostasis and thrombosis. An increased activation of coagulation can result in severe thromboembolic disorders, one of the major reasons responsible for mortality and morbidity in western world. Therefore, an effective, safe, and orally available thrombin inhibitor could be a useful anticoagulant drug for the daily prophylaxis of venous and arterial thrombosis and prevention of myocardial infarction for high-risk patients. Synthetic thrombin inhibitors have a long history; initial compounds were derived from electrophilic ketone- and aldehyde-analogs of arginine. First potent leads of non-covalent inhibitors were developed in the early eighties, which were further optimised in the nineties, after the X-ray structure of thrombin became available. In the meantime a huge number of highly active and selective inhibitors has been published, however, only a few of them have an appropriate pharmacokinetic and pharmacodynamic overall profile, which could justify their further development. Very recently, with Ximelagatran a first orally available thrombin inhibitor has been approved in France for the prevention of venous thromboembolic events in major orthopaedic surgery after successful clinical phase III. However, it still has to be awaited, whether the extensive clinical use of Ximelagatran can demonstrate for the first time that direct thrombin inhibitors offer a real benefit in terms of efficacy and safety over established antithrombotic therapies. This review summarizes the current status of synthetic thrombin inhibitors with a focus on more recently published and promising new compounds.

Plasminogen activator inhibitor-1 (PAI-1) is an important component of the plasminogen / plasmin system as it is the main inhibitor of tissue-type and urokinase-type plasminogen activator. Consequently, PAI- 1 plays an important role in cardiovascular diseases (mainly through inhibition of t-PA) and in cell migration and tumor development (mainly through inhibition of u-PA). As a member of the serpin superfamily, PAI-1 shares important structural properties with other serpins. However, PAI-1 also exhibits unique conformational and functional properties. The current paper provides an overview of the knowledge on PAI-1 gathered since its discovery two decades ago. We are discussing (a) its structural properties and their subsequent association with the functional properties, (b) its role in a wide variety of (patho)physiological processes and (c) a number of strategies to interfere with its functional properties eventually aiming at pharmacological modulation of this risk factor.

The coagulation system is a potent mechanism that prevents blood loss after vascular injury. It consists of a number of linked enzymatic reactions resulting in thrombin generation. Thrombin converts soluble fibrinogen into a fibrin clot. The clot is subsequently removed by the fibrinolytic system upon wound healing. Thrombin-activatable fibrinolysis inhibitor (TAFI), which is identical to the previously identified proteins procarboxypeptidase B, R, and U, forms a link between blood coagulation and fibrinolysis. TAFI circulates as an inactive proenzyme in the bloodstream, and becomes activated during blood clotting. The active form, TAFIa, inhibits fibrinolysis by cleaving off C-terminal lysine residues from partially degraded fibrin that stimulates the tissue-type plasminogen activator-mediated conversion of plasminogen to plasmin. Consequently, removal of these lysines leads to less plasmin formation and subsequently to protection of the fibrin clot from break down. Moreover, TAFI may also play a role in other processes such as, inflammation and tissue repair. In this review, recent developments in TAFI research are discussed.

The natural polyamines spermine, spermidine and putrescine, polycationic molecules at physiological pH, interact with mitochondrial membranes at two specific binding sites exhibiting low affinity and high binding capacity. This binding represents the first step in the electrophoretic mechanism of polyamine transport into mitochondria. Spermine accumulated into the mitochondrial matrix is able to flow out by an electroneutral mechanism. This process promotes bi-directional transport of polyamines in and out of mitochondria, driven by electrical potential and pH gradient, respectively. Polyamines and biogenic amines are oxidized by cytosolic and mitochondrial amine oxidases with the production of hydrogen peroxide and aldehydes, both of which are involved in the induction and / or amplification of the mitochondrial permeability transition (MPT). This phenomenon, which provokes a bioenergetic collapse and redox catastrophe, is strongly inhibited by polyamines in isolated mitochondria. Monoamines also exhibit an inhibitory effect at higher concentrations, but at low concentrations behave as inducer agents. MPT is characterized by the opening of a channel, the transition pore, which permits nonspecific bi-directional traffic of solutes across the inner membrane, leading to swelling of the organelle and release of cytochrome c and apoptosis-inducing factors. These proteins in turn activate the caspase-cascade, which triggers the apoptotic pathway. Depending on their cytosolic concentration, metabolic conditions and cell type, polyamines act as promoting, modulating or protective agents in mitochondrial-mediated apoptosis. While their protective effect could reflect inhibition of MPT and retention of cytochrome c, the promoting effect can be explained by the generation of reactive oxygen species that induce the opposite effect on MPT and cytochrome c release. Polyamines and other active amines can also participate in the regulation of apoptotic pathways by interacting with the mitochondrial tyrosine phosphorylation / dephosphorylation system. Future studies of the multifaceted interactions of polyamines with mitochondria will thus have a substantial impact on our understanding of the physiology of cell proliferation death at several mechanistic levels.

The vast majority of biologically active compounds will never be considered as potential drugs due to inherently poor bioavailability. This review discusses the progress in the development of chemical systems to improve the metabolic stability, absorption and physicochemical properties of potential drugs. Delivery systems that involve the conjugation of lipid and / or sugar moieties are highlighted, as well as novel methods of conjugation of these groups to drugs. The use of sugar molecules to target drugs to particular organs or cells is also discussed, as is the use of lipids in the growing area of gene delivery. This is an update of a previous review [1].