Class Of Direct Thrombin Inhibitors Research Articles

Reversible covalent direct thrombin inhibitors.

IntroductionIn recent years, the traditional treatments for thrombotic diseases, heparin and warfarin, are increasingly being replaced by novel oral anticoagulants offering convenient dosing regimens, more predictable anticoagulant responses, and less frequent monitoring. However, these drugs can be contraindicated for some patients and, in particular, their bleeding liability remains high.MethodsWe have developed a new class of direct thrombin inhibitors (VE-DTIs) and have utilized kinetics, biochemical, and X-ray structural studies to characterize the mechanism of action and in vitro pharmacology of an exemplary compound from this class, Compound 1.ResultsWe demonstrate that Compound 1, an exemplary VE-DTI, acts through reversible covalent inhibition. Compound 1 inhibits thrombin by transiently acylating the active site S195 with high potency and significant selectivity over other trypsin-like serine proteases. The compound inhibits the binding of a peptide substrate with both clot-bound and free thrombin with nanomolar potency. Compound 1 is a low micromolar inhibitor of thrombin activity against endogenous substrates such as fibrinogen and a nanomolar inhibitor of the activation of protein C and thrombin-activatable fibrinolysis inhibitor. In the thrombin generation assay, Compound 1 inhibits thrombin generation with low micromolar potency but does not increase the lag time for thrombin formation. In addition, Compound 1 showed weak inhibition of clotting in PT and aPTT assays consistent with its distinctive profile in the thrombin generation assay.ConclusionCompound 1, while maintaining strong potency comparable to the current DTIs, has a distinct mechanism of action which produces a differentiating pharmacological profile. Acting through reversible covalent inhibition, these direct thrombin inhibitors could lead to new anticoagulants with better combined efficacy and bleeding profiles.

Novel Class of Direct Thrombin Inhibitors Prevent Thrombosis By Inhibiting Fibrinogen Cleavage While Preserving Platelet Function

We have developed a new class of direct thrombin inhibitors (VE-DTIs) that act through reversible covalent enzyme inhibition. These compounds inhibit thrombin with high potency (IC50 < 10 nM) and with better than 100× selectivity against related serine proteases. In rodents, VE-DTIs show dose-dependent efficacy in preventing arterial and venous thromboses respectively in arteriovenous shunt thrombosis and venous stasis thrombosis models. They also prevent thrombin-induced pulmonary embolism in rodents. Despite their strong anticoagulation properties, VE-DTIs are associated with significantly lower bleeding times than other approved anticoagulants dabigatran, argatroban or apixaban in rodent bleeding models (tail bleeding time and saphenous vein bleeding time).We compared the effect of VE-DTIs and the DTIs argatroban and dabigatran on the coagulation cascade. In vitro, VE-DTIs inhibited thrombin-mediated fibrinogen cleavage with similar potency. Inhibition of thrombin/thrombomodulin complex-mediated activation of TAFI and protein C by VE-DTIs were also comparable. In contrast, VE-DTIs were more than 1000-fold less potent in inhibiting thrombin-mediated platelet activation than the other DTIs. Additionally, and also in contrast to argatroban and dabigatran, VE-DTIs protect rodents from the effects of thrombin-induced pulmonary embolism without significantly inhibiting platelet activation. The ability of VE-DTIs to prevent thrombosis without significantly impacting platelet function could lead to a new anticoagulant therapy with lower bleeding risk. DisclosuresSivaraja:Verseon Corporation: Employment, Equity Ownership. Sizikov:Verseon Corporation: Employment, Equity Ownership. Sandoval:Verseon Corporation: Employment, Equity Ownership. Lin:Verseon Corporation: Employment, Equity Ownership. Igoudin:Verseon Corporation: Employment, Equity Ownership. Dash:Verseon Corporation: Employment, Equity Ownership. Xu:Verseon Corporation: Employment, Equity Ownership. Clemens:Verseon Corporation: Employment, Equity Ownership. Estiarte:Verseon Corporation: Employment, Equity Ownership. Shiau:Verseon Corporation: Employment, Equity Ownership. Short:Verseon Corporation: Employment, Equity Ownership. Williams:Verseon Corporation: Employment, Equity Ownership. Datta:Verseon Corporation: Employment, Equity Ownership. Kita:Verseon Corporation: Employment, Equity Ownership.

Production of Thrombin Complexes with DNA Aptamers Containing G-Quadruplex and Different Duplexes

Despite major advances in the understanding of the molecular mechanisms of thrombosis and the development of effective thrombolytic agents, arterial thrombosis remains a formidable clinical problem. A new class of direct thrombin inhibitors (aptamers), has been described recently. Aptamers, single-stranded oligonucleotides with a length of 30-60 nucleotides, exhibit high affinity and specificity towards any defined recognition target (protein). Aptamers are antibody analogs in terms of both specificity and affinity, with an apparent advantage of the former to being reproduced by anautomated chemical synthesis. Aptamers are routinely selected by SELEX technology (Systematic Evolution of Ligands by Exponential enrichment), which is thoroughly discussed elsewhere. The DNA aptamers to thrombin have a very highly ordered tertiary structure (G-quadruplex). A great number of the DNA aptamers to thrombin with sophisticated structure have been designed and widely investigated as potential thrombin inhibitors using traditionalmedicine tests (TT, PT, APPT). In this manuscript we suggest the different structures of aptamers, based on RE31 structure, so-called G-quadruplex, which are bound to short and long duplex. The RE31 aptamer had being previously shown to have an order of magnitude prolonged thrombintime in comparison to 15TBA aptamer. The purpose of the present study was to investigate the trunked aptamers to thrombin structures by CD spectroscopy and estimate the stability of the thrombin complexes with aptamers using electrophoresis in polyacrilamide gels. Our findings clearly show that both G-quadruplex and duplex domains of RE31 as well as trunked aptamers are strong effectors of aptamer complex stability. Using a set of oligonucleotide models derived from RE31 sequence, we have shown that the attached duplex domain of trunked aptamers retains the antiparallel unimolecular G-quadruplex topology seen for 15TBA.

Dabigatran in atrial fibrillation: pharmacology and clinical trials

The central pharmacologic approach to stroke prevention in atrial fibrillation has recently changed with the approval of dabigatran by the US Food and Drug Administration (FDA). Dabigatran is an oral anticoagulant that belongs to the class of direct thrombin inhibitors. Dabigatran has predictable pharmacokinetics, without significant drug and food interactions, rapid onset, and requires twice-daily administration without the need for monitoring. The only drug contraindicated with dabigatran is rifampin. In the Randomized Evaluation of Long-Term Anticoagulation Therapy (RE-LY) trial, dabigatran at a dose of 150mg bid is statistically superior to warfarin in preventing strokes and systemic embolism in patients with atrial fibrillation and has a lower non-statistically significant rate of major bleeding. There is significantly lower rate of intracranial bleeding. The FDA recently approved the 150-mg bid dose for patients with a creatinine clearance above 30mL/min and 75mg bid for use in patients with a creatinine clearance of 15 to 30mL/min. A prespecified subanalysis in both warfarin-experienced and warfarin-naive subgroups mirrored the main results. For cardioversions, a post hoc analysis showed that the rate of thromboembolism and major bleeding within 30days of cardioversion for dabigatran 150mg bid was low and comparable to that of warfarin, with or without transesophageal echocardiography guidance. Dabigatran, therefore, is the first novel anticoagulant to offer an alternative to warfarin.

Bivalirudin: a review of the pharmacology and clinical application

Among the current agents in the class of direct thrombin inhibitors, bivalirudin (Angiomax®, The Medicines Company, NJ, USA) has seen increased use in cardiovascular medicine over the past decade through its primary indication as an anticoagulant used during percutaneous coronary interventions. Bivalirudin has been further investigated and used as the anticoagulation strategy in the setting of cardiac and endovascular surgical procedures and is frequently utilized in the management of patients with heparin-induced thrombocytopenia. In comparison with heparin, bivalirudin exhibits a low immunogenic profile and provides similar or reduced major bleeding rates as well as a predictable degree of anticoagulation that is dose related. Bivalirudin primarily undergoes dual elimination via proteolytic cleavage and renal elimination, and requires dose adjustment in the setting of severe renal dysfunction. Given the body of supportive data, bivalirudin is likely to continue to figure prominently as a reliable and efficient anticoagulation strategy. Additional agents in the class of direct thrombin inhibitors are under investigation and may find increasing clinical use.

New options with dabigatran etexilate in anticoagulant therapy.

Thrombosis, the localized clotting of blood, occurs in both the arterial and venous circulation, and has a major impact on health outcomes. The primary etiology of myocardial infarctions, and approximately 80% of strokes, is acute arterial thrombosis. In combination this represents the most common cause of death in the Western world, while the third leading cause of cardiovascular-associated death is venous thromboembolism. An understanding of the pathogenic changes in the vessel wall and the blood that result in thrombosis is crucial for developing safer and more effective antithrombotic drugs. Dabigatran etexilate belongs to a new class of direct thrombin inhibitors. Following oral administration, dabigatran reaches peak plasma concentrations within 2 hours, shows linear pharmacokinetics, and a limited (but important) amount of direct drug interactions. Given once daily at 150 mg or 220 mg, it has proven to be competitive with enoxaparin in the prevention of venous thromboembolism after major orthopedic surgery, with a comparable safety profile. For stroke prevention in patients suffering from atrial fibrillation, dabigatran administered at a dose of 110 mg twice daily was associated with rates of stroke and systemic embolism that were similar to those associated with warfarin, as well as lower rates of hemorrhage. Dabigatran given at a dose of 150 mg twice daily, as compared with warfarin, was associated with lower rates of stroke and systemic embolism but similar rates of major hemorrhage. Oral bioavailability of dabigatran, together with a rapid onset and offset of action and predictable anticoagulation response, makes this newly available antithrombotic drug an attractive alternative to traditional anticoagulant therapies for numerous thrombosis-related indications.

Ximelagatran increases membrane fluidity and changes membrane lipid composition in primary human hepatocytes

Ximelagatran, the first oral agent in the new class of direct thrombin inhibitors, was withdrawn from the market due to a potential risk of severe liver injury. Increased rates of liver enzyme elevations had been observed during clinical trials of chronic use. Despite intensive preclinical investigations the cellular mechanisms behind the observed hepatic effects remain unknown. The aim of this study was to investigate whether ximelagatran has an effect on the plasma membrane fluidity and the membrane lipid composition which may be important for the cell integrity. After 1 h exposure of primary human hepatocytes with 10 or 100 μM ximelagatran, a significant elevation of membrane fluidity was observed. This elevation was maintained at 24 h, but diminished at 48 h exposure. As changed membrane lipid composition could influence membrane fluidities, changes in membrane lipid profiles were also studied. After 1 h exposure, the phosphatidylcholine/phosphatidylethanolamine molar ratio decreased, whereas the total cholesterol/phospholipid molar ratio decreased after a 48 h exposure. The change in membrane fluidity and lipid composition in human hepatocytes exposed to ximelagatran might indicate changes in plasma membrane properties that in susceptible subjects, could result in loss of membrane integrity and leakage of cellular proteins.

Investigations into the liver effects of ximelagatran using high content screening of primary human hepatocyte cultures

Background: Ximelagatran, the first oral agent in the new class of direct thrombin inhibitors, was withdrawn from the market due to increased rates of liver enzyme elevations in long-term treatments. Despite intensive pre clinical investigations the cellular mechanisms behind the observed hepatic effects remain unknown. Objective: The aim of this study was to assess drug-induced cytotoxicity in primary human hepatocyte cultures by ximelagatran and other reference pharmaceutical agents with known in vivo hepatotoxic profiles. Methods: Drugs cause liver injury by many distinct mechanisms that result in abnormal cellular functioning and different patterns of injury. To address many potential toxic mechanisms in a human-relevant model, freshly isolated human hepatocytes were used in automated imaging assays. Ximelagatran was used as a test compound to study biochemical and morphological changes in human hepatocytes. In addition, 11 control, reference and comparator compounds with known liver-toxic potential in humans were used. The response to these compounds was assessed across five different hepatocyte donor preparations. Results: Cytotoxicity induced by a number of compounds was quantitatively monitored using an automated imaging technique. A variety of morphological changes in hepatocyte cytoskeleton and mitochondrial function could be identified at sublethal doses of test compounds. Doses of ximelagatran up to 500 μM did not cause a cytotoxic response in the majority of preparations and no subcytotoxic response was observed at doses below 125 μM. Conclusions: The experiments described here demonstrate that primary human hepatocytes may be used in a medium-throughput format for screening using imaging-based assays for the identification of cellular responses. Overall, it is concluded that ximelagatran did not cause a significant decrease in cell viability when incubated for 24 h at considerably higher concentrations than are found in plasma following therapeutic dosing.

Antikoagulation zur Sekundärprävention

Thromboembolic events are a major complication of cardiovascular diseases. Secondary prevention of thromboembolic complications can be achieved by anticoagulation with heparin or vitamin-K-antagonists. A new class of direct thrombin inhibitors has been recently introduced into clinical medicine. Clinical studies have demonstrated theses agents to be comparable in efficacy for prophylaxis of venous thromboembolism. However, the results of ongoing trials are awaited in helping to define the place of direct thrombin inhibitors in secondary prevention. Antiplatelet therapy constitutes the cornerstone in the management of patients with acute coronary syndromes and generally high-risk patients with atherothrombosis. Until recently, long-term antiplatelet therapy for the treatment and prevention of atherothrombotic disease was traditionally limited to aspirin. The availability of the thienopyridines and glycoprotein IIb/IIIa inhibitors represents an important addition to the physician's armamentarium. Due to the increased risk of major bleeding, for each patient the decision of anticoagulation and antiplatelet therapy must be tightly correlated to the individual benefit-risk ratio.

Mechanism of Action of the Oral Direct Thrombin Inhibitor Ximelagatran

Thrombin plays a central role in thrombus formation through its conversion of fibrinogen to fibrin and activation of platelets as well as amplifying its own generation by feedback activation via factors V, VIII, and XI. Consequently, thrombin represents a logical and promising target for therapeutic interventions against arterial and venous thromboembolic disorders. Ximelagatran is the first oral agent in the new class of direct thrombin inhibitors and is rapidly absorbed and bioconverted to the active moiety, melagatran, which inhibits fluid-phase and clot-bound thrombin with similar high potency. Binding to the active site of thrombin is direct and competitive and does not require the presence of co-factors. Inhibition of thrombin generation and platelet activation has been demonstrated in vitro with melagatran as well as ex vivo after oral administration of ximelagatran to healthy human volunteers. Oral ximelagatran dose dependently reduced the total thrombus area in an ex vivo flow chamber model of arterial thrombosis, reflecting the cumulative effect of inhibition of thrombin activity, thrombin generation, and platelet activation. Melagatran has also been shown to reduce thrombin-mediated inflammation in vitro. The combination of antithrombotic and anti-inflammatory activity with the practicality of oral dosing provided by ximelagatran represents an important new option for the treatment of arterial and venous thromboembolic disorders.

Prevention of Cardiovascular Events after Acute Coronary Syndrome

Given the pivotal role of thrombin in the pathogenesis of acute coronary syndromes (ACS) and its persistent activation at the site of arterial lesions, antithrombin agents are essential for the prevention of coronary events. Antiplatelet agents are used routinely in the prevention of ACS, but their inability to prevent thrombin generation might contribute to the remaining high rates of recurrent ischemic events after intense antithrombotic treatment in the acute phase. Combination treatment with antiplatelet agents and anticoagulants, such as low-molecular-weight heparins (LMWH) and vitamin K antagonists, provides improved efficacy in the secondary prevention of ACS but these agents have limitations that prevent widespread adoption of their use for long-term treatment. Ximelagatran is the first oral agent in the new class of direct thrombin inhibitors (DTIs) and has considerable therapeutic potential in ACS. The DTIs are able to inhibit free and fibrin-bound thrombin by directly binding to the thrombin catalytic site. Furthermore, the oral administration and predictable pharmacokinetics of ximelagatran mean that it can be used at a fixed dose without coagulation monitoring and is convenient for long-term therapy. The efficacy of ximelagatran in the prevention of coronary events has been investigated in patients with recent myocardial infarction (MI) in the phase II Efficacy and Safety of the Oral Direct Thrombin inhibitor Ximelagatran in Patients with Recent Myocardial Damage (ESTEEM) trial. Ximelagatran (24 to 60 mg twice daily) added to aspirin (160 mg once daily) reduced the risk of the composite end point of death, MI, and severe recurrent ischemia by 24% versus aspirin alone, with no significant increase in major bleeding. Elevated serum transaminase enzymes developed during the first 1 to 6 months of treatment in a proportion of patients given ximelagatran. These elevations usually abated without clinical sequelae whether or not treatment was continued. The ESTEEM results highlight the potential for ximelagatran as an efficacious and well-tolerated long-term treatment for the prevention of arterial thrombotic events.

Ximelagatran (Exanta): Alternative to Warfarin?

For decades, the standards of antithrombotic therapy have been heparin and coumarin compounds, primarily warfarin (1). Other newer approaches include low-molecular-weight heparins, fondaparinux, lepirudin, argatroban, and melagatran (investigational). Although existing treatments are effective, they have many limitations. A safer, more convenient therapy is needed. Ximelagatran is the first oral treatment in a new World Health Organization class of direct thrombin inhibitors and is the first new oral anticoagulant since the introduction of warfarin almost 60 years ago (2). It was submitted to the Food and Drug Administration (FDA) for approval in December 2003 (3).

A review of the effects of the oral direct thrombin inhibitor ximelagatran on coagulation assays

Ximelagatran is the first oral agent in the new anticoagulant drug class of direct thrombin inhibitors (DTIs)1 and has been clinically developed using fixed dosing without the use of coagulation monitoring. While coagulation monitoring is not required with ximelagatran, effects on various coagulation assays have been studied. This paper describes the effects of melagatran, the active form of ximelagatran, on different coagulation assays and considers their possible use in special clinical situations.

New advances in stroke prevention in atrial fibrillation: ximelagatran and direct thrombin inhibition

Ximelagatran is the first oral agent in a new class of direct thrombin inhibitors and is under investigation for the prevention and treatment of a range of thromboembolic disorders. Atrial. fibrillation (AF) is associated with a markedly increased risk of stroke and, while study results have consistently demonstrated that anticoagulant therapy with warfarin reduces this risk, there are substantial unmet needs in the management of AF-associated thrombotic risk. In particular, warfarin has unpredictable activity leading to the risk of bleeding complications and the need for routine anticoagulant monitoring. Ximelagatran has a range of potential benefits over warfarin in the prevention of AF-associated stroke, including predictable anticoagulant activity that is not affected by food and a tow potential for drug-drug interactions. Consequently, the risk of bleeding complications may be,reduced and there is a fixed-dose regimen with no requirement for routine anticoagulant monitoring. The SPORTIF (Stroke Prevention using an ORal Thrombin Inhibitor in atrial. Fibrillation) series of studies is investigating the extent to which these potential advantages translate into clinical benefits. SPORTIF III is a Phase III study of the efficacy and safety of ximelagatran compared with warfarin in the prevention of stroke in 3407 patients with AF. The results demonstrate that ximelagatran has effectiveness non-inferior to well-controlled warfarin in the prevention of stroke and is associated with a lower risk of bleeding complications. With its predictable anticoagulant effect and the enhanced convenience offered by a fixed-dose regimen and with no requirement for routine coagulation monitoring, these results indicate that ximelagatran promises clinical benefits over warfarin that could improve the management of stroke risk in patients with AF. (C) 2004 The European Society of Cardiology. Published by Elsevier Ltd. All rights reserved. (Less)

Management of Patients with Heparin-Induced Thrombocytopenia During Cardiac Surgery

The role of heparin-induced thrombocytopenia and its possibly severe complications in patients undergoing cardiac surgery is increasingly appreciated. Increasing evidence indicates that in patients without the presence of antibodies, unfractionated heparins can be safely used during cardiopulmonary bypass, if their employment is restricted to this short period. This strategy is the first choice in patients with a current negative status of antibodies and in patients in whom surgery can be delayed until antibodies cannot be detected by laboratory tests. To date, however, no alternative anticoagulant to heparin during cardiopulmonary bypass has been approved. With the introduction of the class of direct thrombin inhibitors, effective anticoagulants have become available; however, they have no antidote and require specific monitoring. The availability of direct thrombin inhibitors is currently restricted. The administration of unfractionated heparin with anti-platelet agents such as prostaglandins or the short-acting platelet glycoprotein llb/Illa antagonist tirofiban is another option. Despite successful use of these strategies in fairly large numbers of patients, recent information suggests that in the case of tirofiban, renal failure may cause persistence of the agent with subsequent severe hemorrhage. If surgery is restricted to coronary artery bypass grafting, recent data show that off-pump strategies with use of the direct thrombin inhibitor bivalirudin appear to be a promising option. Therefore, if surgery cannot be postponed, the anticoagulation protocol and the surgical strategy must be adjusted to the condition of the patient and the experience of the center in order to reduce the risk of these “offlabel” strategies.