piperidines and melagatran

The present study investigated the effect of the thrombin inhibitors antithrombin (AT) (with and without unfractionated heparin or low molecular weight heparin), hirudin, inogatran and melagatran on thrombin-thrombomodulin-mediated generation of activated protein C (APC), in solution and on endothelial cells. Sequential incubation with thrombin, thrombin inhibitors and protein C was followed by measurement of APC by an amidolytic assay. The approximate concentrations resulting in 50% inhibition of endothelial cell-mediated APC generation for AT, AT-unfractionated heparin, AT-low molecular weight heparin, hirudin, melagatran and inogatran were 200, 4, 9, 1, 8 and 60 nmol/l, respectively. The normal plasma level of AT is 2800 nmol/l and relevant therapeutic concentrations from clinical trials are 200 nmol/l for hirudin, 500 nmol/l for melagatran and 1000 nmol/l for inogatran. The present study indicates that clinically relevant concentrations of the tested thrombin inhibitors interfere with endothelial-mediated APC generation, which may offer an explanation for the lack of a dose-response effect in clinical trials with thrombin inhibitors.

Topics: Antithrombins; Azetidines; Benzylamines; Dose-Response Relationship, Drug; Endothelium, Vascular; Flow Cytometry; Glycine; Heparin; Hirudins; Humans; Piperidines; Protein C; Thrombin; Thrombomodulin

Thrombin-induced platelet activation involves cleavage of protease-activated receptors (PARs) 1 and 4, and interaction, via glycoprotein (Gp)Ibalpha, with the platelet GpIb/IX/V complex. This study investigated inhibition of platelet activation by thrombin inhibitors with different modes of action: two reversible direct thrombin inhibitors, melagatran and inogatran; hirudin, a tightly binding direct thrombin inhibitor; and two indirect thrombin inhibitors, heparin and dalteparin. Up-regulation of P-selectin (CD62P) and PAR-1 cleavage was measured in human whole blood, by flow cytometry. The thrombin concentration that induced 50% of maximum (EC50 ) PAR-1 cleavage was 0.028 nmol/l, while that of platelet activation (CD62P) was over two-fold higher (0.64 nmol/l). The EC50 of a PAR-1-independent component, defined as a further activating effect of thrombin on top of the maximum PAR-1-activating peptide (AP) effect, was 3.2 nmol/l. All anticoagulants were concentration-dependent inhibitors of thrombin-induced platelet activation and PAR-1 cleavage, but none inhibited PAR-1-AP or PAR-4-AP induced activation. Melagatran and inogatran were more potent inhibitors of CD62P up-regulation than of PAR-1 cleavage; conversely, hirudin, heparin and dalteparin were more potent inhibitors of PAR-1 cleavage.Thus, reversible direct thrombin inhibitors, such as melagatran, are potent inhibitors of thrombin-induced platelet activation, acting mainly by inhibition of a PAR-1-independent component.

Topics: Adult; Anticoagulants; Azetidines; Benzylamines; Dalteparin; Dose-Response Relationship, Drug; Flow Cytometry; Glycine; Heparin; Hirudins; Humans; Male; P-Selectin; Piperidines; Platelet Activation; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIb-IX Complex; Receptor, PAR-1; Receptors, Thrombin; Statistics as Topic; Thrombin

Clinical trials evaluating direct thrombin inhibitors in unstable coronary artery disease (CAD) have been disappointing. The hypothesis tested in the present study was that these agents may inhibit the anticoagulant effect of thrombin to a further extent than the procoagulant effect of thrombin.. We studied both reversible and irreversible thrombin inhibitors and compared the effects of each inhibitor on activated protein C (APC) generation vs. the effect on fibrinopeptide A (FPA) generation. A mixture of protein C, thrombin inhibitor, fibrinogen, fibrin polymerisation blocker and thrombin was incubated with thrombomodulin (TM)-expressing human saphenous vein endothelial cells (HSVECs). The inhibitors investigated were melagatran, inogatran, hirudin, hirugen, D-Phe-D-Pro-D-arginyl chloromethyl ketone (PPACK), and antithrombin (AT) alone or in combination with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH).. All agents, except hirugen, inhibited APC and FPA generation in a dose-dependent manner. FPA inhibition/APC inhibition ratios, based on IC50 for inogatran, melagatran, hirudin, PPACK, AT, AT-UFH and AT-LMWH were 1.73, 0.85, 0.55, 2.1, 0.5, 0.65 and 3.1 respectively.. All agents, except hirugen, inhibited APC and FPA generation approximately to a similar extent. Thus, it can be inferred that the poor efficacy of thrombin inhibitors in recent clinical trials in patients with unstable CAD is unlikely to be a consequence of their effects on the protein C system.

Topics: Amino Acid Chloromethyl Ketones; Anticoagulants; Azetidines; Benzylamines; Clinical Trials as Topic; Coagulants; Coronary Artery Disease; Fibrinopeptide A; Glycine; Hirudins; Humans; Peptide Fragments; Piperidines; Protein C; Thrombin

Oral thrombin inhibitors are under development as potential drugs for prophylaxis and treatment of thrombotic events. The effect of pretreatment with two direct thrombin inhibitors, melagatran and inogatran, was evaluated in a rat model of cerebral infarction. Ischaemic stroke was induced by photochemical reaction after an injection of Rose Bengal and focused posterior and to the right of the intersection of the coronal and sagittal sutures on the intact calvarium. A single oral dose of melagatran (30 micromol/kg) significantly reduced the volume of the cortical infarct by 53% (P<.05) compared with control. In addition, following intravenous inogatran (6 micromol/kg) or oral inogatran (100 micromol/kg), the volume of the cortical infarct decreased by 83% and 19%, respectively, compared with control. This study showed that experimental focal ischaemic infarction, elicited by photochemically induced endothelial cell damage, can be significantly reduced with melagatran and inogatran, direct thrombin inhibitors.

Topics: Administration, Oral; Animals; Antithrombins; Azetidines; Benzylamines; Brain Infarction; Cerebral Cortex; Drug Evaluation, Preclinical; Glycine; Hypertension; Injections, Intravenous; Light; Male; Photochemistry; Piperidines; Rats; Rats, Inbred SHR

The thrombin inhibitors argatroban, efegatran, NAPAP, CH 1091, CH 248, inogatran and melagatran have been characterised with respect to their mechanism of binding to human alpha-thrombin. Stopped-flow spectrophotometry was used to follow thrombin-catalysed hydrolysis of the chromogenic substrate S-2238 in the presence of inhibitors. The rate of onset or decay of inhibition was evaluated using progress curve analysis. It was possible to obtain apparent association and dissociation rate constants from the dependence of the rates on the inhibitor concentrations. Inhibition constants calculated from the association and dissociation rate constants were in good agreement with those calculated from steady-state rates. The binding of 6 inhibitors was also monitored directly using stopped-flow spectrofluorimetry when two kinetic components were found with all inhibitors. The faster component accounted for the largest part of the change in the intrinsic fluorescence of thrombin induced by inhibitor binding and was dependent on the inhibitor concentration. The slower component was independent of the concentration of the inhibitor. The concentration dependence of the faster component was linear with the compounds argatroban, NAPAP, CH 1091 and melagatran and hyperbolic with the compounds CH 248 and inogatran. The values of the apparent second-order rate constants at pH 7.4 and 37 degrees C range from slow to rapid binding in the interval 16-78 x 10(6) M-1 s-1, which is somewhat higher than 1-34 x 10(6)M-1 s-1 obtained from progress curve analysis of the onset of inhibition. The present results support a mechanism that includes rearrangement of a weak initial thrombin-inhibitor complex towards a tighter complex. Moreover, at least one additional step is required in the mechanism. In this model, the rate-limiting step for the binding of the inhibitor at concentrations in the nanomolar range depends on the primary interaction between the inhibitor and native thrombin.

Topics: Antithrombins; Area Under Curve; Arginine; Azetidines; Benzylamines; Binding Sites; Dipeptides; Fluorescence; Glycine; Humans; Kinetics; Molecular Weight; Pipecolic Acids; Piperidines; Spectrometry, Fluorescence; Spectrophotometry; Sulfonamides; Thrombin

The relation between the antithrombotic effect in vivo, and the inhibition constant (Ki) and the association rate constant (k(on)) in vitro was investigated for eight different thrombin inhibitors. The carotid arteries of anaesthetized rats were exposed to FeCl3 for 1 h, and the thrombus size was determined from the amount of incorporated 125I-fibrinogen. The thrombin inhibitors were given intravenously, and complete concentration- and/or dose-response curves were constructed. Despite a 50,000-fold difference between the Ki-values comparable plasma concentrations of hirudin and melagatran were needed (0.14 and 0.12 micromol l(-1), respectively) to obtain a 50% antithrombotic effect (IC50) in vivo. In contrast, there was a comparable in vitro (Ki-value) and in vivo (IC50) potency ratio for melagatran and inogatran, respectively. These results can be explained by the concentration of thrombin in the thrombus and improved inhibition by the low-molecular-weight compounds. For all eight thrombin inhibitors tested, there was an inverse relationship between k(on)-values in vitro and the slope of the dose response curves in vivo. Inhibitors with k(on)-values of < 1 x 10(7) M(-1) s(-1) gave steep dose response curves with a Hill coefficient > 1. The association time for inhibition of thrombin for slow-binding inhibitors will be too long to give effective antithrombotic effects at low plasma concentrations, but at increasing concentrations the association time will decrease, resulting in a steeper dose-response curve and thereby a more narrow therapeutic interval.

Topics: Amino Acid Chloromethyl Ketones; Animals; Anticoagulants; Arginine; Azetidines; Benzylamines; Carotid Artery Thrombosis; Dose-Response Relationship, Drug; Fibrinolytic Agents; Glycine; Hemodynamics; Heparin; Hirudin Therapy; Hirudins; Kinetics; Male; Oligopeptides; Partial Thromboplastin Time; Peptide Fragments; Pipecolic Acids; Piperidines; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Sulfonamides; Thrombin; Thrombin Time

A sensitive thrombosis model with a high reproducibility was developed in the rat, utilizing stasis of the caval vein and a standardized surgical trauma as the only thrombogenic stimuli. Since no procoagulant substances were used, the results of the present study might be relevant in a clinical situation. The antithrombotic effect of two recently synthesized low-molecular-weight thrombin inhibitors have been compared to dalteparin, (Fragmin) a low-molecular-weight heparin fragment. Each compound was studied at 8 different doses with 10 rats in each group. On a gravimetric basis, the thrombin inhibitor melagatran was twice as potent as dalteparin (ED50 16 and 33 microg/kg per h, respectively). The second thrombin inhibitor, inogatran, had an intermediate effect, with an ED50 of 24 microg/kg per h. No differences in antithrombotic effect were, however, found when the compounds were compared at anticoagulant equivalent doses (same APTT prolongation). A 50% reduction in the mean thrombus weight was obtained when APTT was prolonged to 1.2 to 1.3 times the pretreatment value.

Topics: Animals; Azetidines; Benzylamines; Dalteparin; Disease Models, Animal; Fibrinolytic Agents; Glycine; Heparin, Low-Molecular-Weight; Infusions, Intravenous; Injections, Intravenous; Male; Piperidines; Rats; Rats, Wistar; Thrombin; Thrombophlebitis; Vena Cava, Inferior