Dabigatran aggravates topoisomerase I peptide-loaded dendritic cells-induced lung and skin fibrosis.

OBJECTIVES: Dysregulated coagulation cascade has been implicated in development of fibrosis in systemic sclerosis (SSc). Thrombin, a key mediator of the coagulation pathway, has both proinflammatory and procoagulant properties. Here, we evaluated the efficacy of oral dabigatran, a direct thrombin inhibitor, on topoisomerase I dendritic cells (TOPOIA DCs)-induced lung and skin fibrosis, an experimental model of SSc. METHODS: Mice were repeatedly immunized with TOPOIA DCs. Dabigatran was administered in food either during the onset of fibrotic (late treatment) or inflammatory (early treatment) phase. RESULTS: Early administration of dabigatran caused an aggravation of pulmonary fibrosis associated with signs of severe perivascular inflammation while late treatment was not protective when compared to the untreated TOPOIA DCs group. Thrombin was increased in lungs of TOPOIA DCs immunized group and, paradoxically, further augmented by administration of dabigatran to immunized mice. As in lungs, early and not late drug administration exacerbated skin fibrosis. Moreover, early dabigatran treatment induced a profibrotic and inflammatory skin gene expression signature with upregulated expression of Col5a1, Timp1, Tweakr, Vwf, Il6, Il33, Il4 and Ifng. CONCLUSIONS: Dabigatran aggravated lung and skin fibrosis in a TOPOIA DCs-induced model of SSc-like disease. Therefore, our results argue against the use of dabigatran to treat patients with SSc.

Design, Synthesis, and Biological Evaluation of Dabigatran Etexilate Mimics, a Novel Class of Thrombin Inhibitors.

Human α-thrombin is a particularly promising target for anticoagulant therapy, and identification of oral small-molecular inhibitors of thrombin remains a research focus. On the basis of the X-ray crystal structure of human α-thrombin and its inhibitor dabigatran, we designed and synthesized a series of dabigatran etexilate mimics containing a novel tricyclic fused scaffold. The biological evaluations reveal that all of the compounds possess moderate activity of antiplatelet aggregation induced by thrombin in vitro. Moreover, compound I-8, which contains 2-hydroxymethyl-3,5,6-trimethylpyrazine (HTMP), a cleavable moiety with antiplatelet activity, shows the best anticoagulant effect among the tested compounds in vivo. Those synthesized compounds that have better in vitro activity were subjected to bleeding complication tests, and the results demonstrate that the novel compounds are less likely to have bleeding risk than dabigatran etexilate.

Two-year carcinogenicity studies with the oral direct thrombin inhibitor ximelagatran in the rat and the mouse.

The results of 18 months mouse and 24 months rat carcinogenicity studies with the oral direct thrombin inhibitor ximelagatran are presented. In the mouse, gavage doses of ximelagatran up to 180 µmol/kg per d produced no neoplastic changes in any of the tissues examined. In the rat, gavage doses up to 240 µmol/kg per d produced multiple macroscopically detectable nodules in the pancreas, which are seen to be focal/multifocal acinar cell hyperplasia and focal/multifocal acinar cell adenoma upon histological evaluation. There were no other treatment-related effects on tumor incidence or distribution in the rat. The studies show a clear species difference in pancreatic effects between the rat and the mouse to long-term treatment with ximelagatran.

Vascular protease-activated receptor 4 upregulation, increased platelet aggregation, and coronary lipid deposits induced by long-term dabigatran administration - results from a diabetes animal model.

Essentials The impact of long-term thrombin inhibition outside the coagulation cascade is far from clear. We aimed to assess the impact of dabigatran etexilate (DE) in diabetic and control rats. In diabetic rats, DE increased platelet aggregation and lead to coronary lipid deposits. Long-term thrombin inhibition may increase atherosclerotic and atherothrombotic risk. SUMMARY: Background Besides its role in the coagulation cascade, thrombin contributes to platelet aggregation and to a plethora of non-hemostatic functions. Objectives To assess the impact of long-term thrombin inhibition with dabigatran etexilate (DE) on platelet aggregation and on extrahemostatic thrombin-related functions in diabetic and control rats. Methods Markers of inflammation, endothelial dysfunction, oxidative stress, angiogenesis and cell adhesion molecules were quantified in control rats (Control; n = 6), DE-treated control rats (Control-Dabi; n = 8), diabetic rats (Diabetes; n = 5), and DE-treated diabetic rats (Diabetes-Dabi; n = 8). Agonist-induced platelet aggregation, aortic and coronary lipid deposits and aortic protease-activated receptor 4 (PAR4) expression were also assessed. Results Control-Dabi rats showed significantly higher high-sensitivity C-reactive protein, von Willebrand factor (VWF), vascular endothelial growth factor (VEGF) and fibronectin levels, and significantly lower PAR4 agonist-induced aggregation, than Control rats. Control-Dabi rats also showed mild aortic lipid deposits, whereas no such changes were observed in Control rats. Diabetes-Dabi rats showed significantly higher VWF, VEGF and fibronectin levels than Diabetes rats, and similar PAR4 agonist-induced aggregation as Diabetes rats, and significantly higher ADP-induced aggregation than Diabetes rats. Coronary lipid deposits were observed in 75% of Diabetes-Dabi rats and in none of the Diabetes rats. PAR4 expression was 20.4% higher in Control-Dabi rats and 27.4% higher in Diabetes-Dabi rats than in their non-treated peers. Conclusions This study indicates that long-term thrombin inhibition increases vascular PAR4 expression, promotes atherosclerosis-related mechanisms, and, in diabetic rats, increases platelet aggregation and favors the occurrence of coronary lipid deposits. These experimental data suggest that long-term thrombin inhibition may increase atherosclerotic and atherothrombotic risk, particularly in the presence of diabetes.

Dose requirements for idarucizumab reversal of dabigatran in a lethal porcine trauma model with continuous bleeding.

Idarucizumab is licensed for emergency reversal of dabigatran. A single 5 g dose is usually sufficient, but higher doses may sometimes be required and optimum dosing has not been defined. It was the aim of this study to investigate the effect of idarucizumab, given once or as a split dose, after double trauma in pigs anticoagulated with dabigatran. Dabigatran etexilate (30 mg/kg bid) was given to 18 male pigs orally for 3 days. On day 4, animals were randomised 1:1:1 to receive idarucizumab 60+0, 60+60 or 120+0 mg/kg. Doses were administered 15 and 75 minutes after initial liver trauma. At 60 minutes, a second liver injury was undertaken. Animals were monitored for 5 hours after initial trauma or until death. Blood loss during the first hour was 990 ± 109 ml, 988 ± 84 ml and 964 ± 75 ml in the 60+0, 60+60 and 120+0 groups, respectively. In the 120+0 and 60+60 groups, total blood loss was 1659 ± 346 and 1426 ± 106 ml, respectively, and survival at 5 hours was 100 %. However, in the 60+0 group, total blood loss was 3561 ± 770 ml and survival was 50 %. Analysis of dabigatran plasma concentrations showed that equimolar concentrations of idarucizumab are necessary to bind all dabigatran and achieve sufficient thrombin generation. At sufficient doses, idarucizumab rapidly reduced blood loss and improved survival in this lethal porcine model of double trauma with dabigatran anticoagulation. In clinical practice, should bleeding continue after initial treatment with the approved 5 g dose of idarucizumab, a second dose may potentially be effective to control bleeding caused by redistribution of unbound dabigatran.

A novel hirudin derivative inhibiting thrombin without bleeding for subcutaneous injection.

Currently, anticoagulants would be used to prevent thrombosis. Thrombin is an effector enzyme for haemostasis and thrombosis. We designed a direct thrombin inhibitor peptide (DTIP) using molecular simulation and homology modelling and demonstrated that the C-terminus of DTIP interacts with exosite I, and N-terminus with the activity site of thrombin, respectively. DTIP interfered with thrombin-mediated coagulation in human, rat and mouse plasma (n=10 per group) and blocked clotting in human whole blood in vitro. When administered subcutaneously, DTIP showed potent and dose-dependent extension of aPTT, PT, TT and CT in rats (n=10 per group). The antithrombotic dose of DTIP induced significantly less bleeding than bivalirudin determined by transecting distal tail assay in rats. Furthermore, DTIP reached peak blood concentration in 0.5-1 hour and did not cause increased bleeding after five days of dosing compared to dabigatran etexilate. The antithrombotic effect of DTIP was evaluated in mice using lethal pulmonary thromboembolism model and FeCl3-induced mesenteric arteriole thrombus model. DTIP (1.0 mg/kg, sc) prevented deep venous thrombosis and increased the survival rate associated with pulmonary thromboembolism from 30 % to 80 %. Intravital microscopy showed that DTIP (1.0 mg/kg, sc) decelerated mesenteric arteriole thrombosis caused by FeCl3 injury. These data establish that DTIP is a novel antithrombotic agent that could be used to prevent thrombosis without conferring an increased bleeding risk.

Exploiting the antithrombotic effect of the (pro)thrombin inhibitor bothrojaracin.

Bothrojaracin is a 27 kDa C-type lectin-like protein from Bothrops jararaca snake venom. It behaves as a potent thrombin inhibitor upon high-affinity binding to thrombin exosites. Bothrojaracin also forms a stable complex with prothrombin that can be detected in human plasma. Formation of the zymogen-inhibitor complex severely decreases prothrombin activation and contributes to the anticoagulant activity of bothrojaracin. In the present study, we employed two rodent models to evaluate the antithrombotic effect of bothrojaracin in vivo: stasis-induced thrombosis and thrombin-induced pulmonary thromboembolism. It was observed that bothrojaracin interacts with rat prothrombin in plasma. Ex-vivo assays showed stable complex formation even after 24 h of a single bothrojaracin dose. As a result, bothrojaracin showed significant antithrombotic activity in a rat venous thrombosis model elicited by thromboplastin combined with stasis. The antithrombotic activity of bothrojaracin (1 mg/kg) persisted for up to 24 h and it was associated with moderate bleeding as assessed by a tail transection method. Formation of bothrojaracin-prothrombin complex has been also observed following intravenous administration of the inhibitor into mice. As a result, bothrojaracin effectively protected mice from thrombin-induced fatal thromboembolism. We conclude that bothrojaracin is a potent antithrombotic agent in vivo and may serve as a prototype for the development of new zymogen-directed drugs that could result in prolonged half-life and possible decreased hemorrhagic risk.

Reversal of lipophilic weak bases using pH gradient acidic centre liposomes: demonstration of effect in dabigatran-induced anticoagulation.

INTRODUCTION: Liposomes have recently emerged as rational vehicles for drug detoxification. Modification of the core pH may further enhance the ability of liposomes to sequester lipophilic toxins that are weak bases. Dabigatran, a reversible inhibitor of thrombin, has been widely promoted as a novel oral anticoagulant. As a lipophilic weak-base, it provides a rational target for reversal with acidic-centred liposomal preparations. The present study tests the hypothesis that acidic centre liposomes will reverse dabigatran induced anticoagulation. METHOD: Following enteric dabigatran dosing in vitro assessment of thrombin clotting times (TCT) was undertaken in rabbit plasma spiked with incremental liposome concentrations. Tail vein bleeding was assessed following intravenous liposome injection in rats after enteric dabigatran administration. RESULTS: Liposomes achieved reversal of TCT to baseline at low levels of thrombin inhibition, and partial reversal of TCT at higher levels. Liposomes completely reversed the effects of dabigatran on rat tail vein bleeding time (134.0 (6.7) s liposomes vs. 410 (37.8) s control; p < 0.01). CONCLUSION: Dabigatran-induced coagulopathy was reversed in vitro and in vivo by acidic-centred liposomes. pH-modified liposomes are a promising investigational entity in the antidotal treatment of pharmacologic weak bases that are lipid soluble at physiologic pH.

Evaluation of the toxicology, pharmacokinetics, and local tolerance of recombinant factor IX Fc fusion protein in animals.

INTRODUCTION: Recombinant factor IX Fc fusion protein (rFIXFc) is a recombinant coagulation factor composed of a single molecule of recombinant factor IX (rFIX) covalently fused to the Fc domain of human immunoglobulin G1 (IgG1) with no intervening sequence. An extensive nonclinical program was performed to support the clinical development of rFIXFc for treatment of people with hemophilia B. MATERIALS AND METHODS: Repeat-dose toxicology studies of rFIXFc were performed in 2 relevant species: Sprague Dawley rats (4-week study) and cynomolgus monkeys (5- and 27-week studies). Assessments included in-life observations, electrocardiograms (monkeys only), laboratory evaluations (including hematology and blood chemistry), postmortem analyses, local tolerance, and pharmacokinetics (PK). Allometric scaling was performed with PK data from multiple species, including humans. Local tolerance (single-dose study) and thrombogenic potential (Wessler stasis model) of rFIXFc were tested in New Zealand White rabbits. RESULTS: There were no significant local or systemic toxicity findings in the repeat-dose studies. Allometric scaling data suggested that animal rFIXFc PK results are predictive of human PK parameters. There were no findings from the local tolerance study in rabbits; thrombogenic activity was less than that elicited by rFIX and a prothrombin complex concentrate, and similar to vehicle control. CONCLUSIONS: rFIXFc was well tolerated in toxicology studies and demonstrated a low thrombogenic potential. These results are consistent with phase 1/2a and phase 3 clinical studies of rFIXFc in people with hemophilia B.

Differential Effects of Dabigatran and Warfarin on Bone Volume and Structure in Rats with Normal Renal Function.

BACKGROUND: Warfarin, a widely used anticoagulant, is a vitamin K antagonist impairing the activity of vitamin K-dependent Bone Gla Protein (BGP or Osteocalcin) and Matrix Gla Protein (MGP). Because dabigatran, a new anticoagulant, has no effect on vitamin K metabolism, the aim of this study was to compare the impact of warfarin and dabigatran administration on bone structure and vascular calcification. METHODS: Rats with normal renal function received for 6 weeks warfarin, dabigatran or placebo. Bone was evaluated immuno-histochemically and hystomorphometrically after double labelling with declomycin and calcein. Aorta and iliac arteries were examined histologically. RESULTS: Histomorphometric analysis of femur and vertebrae showed significantly decreased bone volume and increased trabecular separation in rats treated with warfarin. Vertebra analysis showed that the trabecular number was higher in dabigatran treated rats. Osteoblast activity and resorption parameters were similar among groups, except for maximum erosion depth, which was higher in warfarin treated rats, suggesting a higher osteoclastic activity. Therefore, warfarin treatment was also associated with higher bone formation rate/bone surface and activation frequency. Warfarin treatment may cause an increased bone turnover characterized by increased remodelling cycles, with stronger osteoclast activity compared to the other groups. There were no differences among experimental groups in calcium deposition either in aortic or iliac arteries. CONCLUSIONS: These findings suggest for the first time that dabigatran has a better bone safety profile than warfarin, as warfarin treatment affects bone by reducing trabecular size and structure, increasing turnover and reducing mineralization. These differences could potentially result in a lower incidence of fractures in dabigatran treated patients.

Effect of toxic shock syndrome toxin-1 on human hemostatic parameters.

The effect of toxic-shock-syndrome-toxin-1 (TSST-1) on coagulation and platelet aggregation was studied in blood samples from human healthy volunteers. TSST-1 at 5 micrograms/ml does not modify the extrinsic or intrinsic coagulation pathways. However, thrombin clotting time (TCT) was significantly increased in the presence of TSST-1. Platelet aggregation was not directly affected by TSST-1 but the toxin strongly inhibited platelet aggregation induced either by epinephrine, adenosine diphosphate (ADP) or platelet activating factor (PAF), while having no effect on aggregation induced by thrombin, collagen or a calcium ionophore. The above results suggest that TSST-1 may bind to a transducer common for some aggregators or that it may induce some non-specific modification of platelet membrane.

Pharmacological characterization of a new 4-amidinophenyl-alanine thrombin-inhibitor (CRC 220).

The new thrombin inhibitor CRC 220 was characterized in vivo for its antithrombotic effects. CRC 220 led to a dose-dependent prolongation of clotting parameters as determined in rats, rabbits, dogs, sheeps, pigs and monkeys. We evaluated the efficacy of CRC 220 to prevent thrombus formation in arteries and in the microcirculation in different animal models. In a rabbit model of tissue factor-induced coagulation activation, infusion of 0.5 mg/kg x h CRC 220 (3 hours) led to a significant prevention of fibrinogen decrease. In a rat model of lethal LPS-induced DIC CRC 220 significantly prevented the mortality rate after a 4h-infusion of 0.75 mg/kg x h. Thrombin-induced platelet aggregation in rat lungs could be prevented by the i.v. bolus injection of CRC 220. A dose of 0.3 mg/kg leads to a reduction of more than 80% of platelet deposition in the lung, significant inhibition was still observed 90 minutes after CRC 220 administration; at this time the inhibitor had already been cleared from plasma. Arterial thrombosis was induced in rabbits by squeezing and stenosis of the A. carotis. The i.v. bolus administration of CRC 220 dose-dependently prevented thrombus formation, an ED50 of 0.03 mg/kg was calculated. This dose was associated with only a minor prolongation of aPTT.

[Chromogenic substrate as antidote against the thrombin inhibitor Melagatran?]. / Chromogenes Substrat als Antidot gegen den Thrombininhibitor Melagatran?

It could be shown in vitro that a chromogenic substrate (Chromozym TH, Roche Mannheim) acts at least partially as antidote against the new thrombin inhibitor Melagatran (AstraZeneca, Mölndal, Sweden). It is discussed that this antidote effect of a chromogenic substrate might be due to a substrate competition of fibrinogen, thrombin inhibitor, and chromogenic substrate for thrombin. Further animal experiments will clarify whether this in vitro observation is of practical relevance in vivo, too.

Antithrombotic and anticoagulant effects of direct factor Xa inhibitor darexaban in rat and rabbit models of venous thrombosis.

The oral direct factor Xa inhibitor darexaban administered intraduodenally prevented venous thrombus formation in both rats and rabbits with no effect on bleeding. The indirect parenteral Factor Xa inhibitor fondaparinux exerted similar properties, only prolonging bleeding time at extremely high doses. In contrast, the thrombin inhibitor ximelagatran and low-molecular-weight heparin enoxaparin prolonged bleeding time at antithrombotic doses. Studies using human platelets showed darexaban glucuronide, a darexaban metabolite that predominantly determines darexaban antithrombotic effects in vivo, had no effect on platelet activation and aggregation, while heparin and enoxaparin activated platelets. Melagatran, heparin, and enoxaparin all inhibited thrombin-induced platelet aggregation at clinically relevant concentrations. Taken together, these results suggest that thrombin-inhibiting drugs may increase the risk of bleeding, while darexaban may have potential as an orally available antithrombotic agent with a wide therapeutic window.

Reduction of thrombus size in murine models of thrombosis following administration of recombinant α1-proteinase inhibitor mutant proteins.

The variant serpin α1-PI M358R inhibits thrombin and other proteases such as activated protein C (APC) and factor XIa. We previously described recombinant proteins HAPI M358R (α1-PI M358R containing an N-terminal extension corresponding to residues 1-75 of heparin cofactor II) and HAPI RCL5 (HAPI M358R with F352-I356 and I360 substituted for the corresponding residues of antithrombin), with enhanced selectivity for thrombin over APC inhibition. We tested the hypotheses that these recombinant proteins would limit thrombosis in three mouse models, and that the HAPI chimeric proteins would be more effective than α1-PI M358R. Recombinant serpins were purified from Escherichia coli by nickel chelate and ion exchange affinity chromatography, and administered to mice intravenously. HAPI RCL5 reduced incorporation of radiolabelled fibrin(ogen) into thrombi in the ferric chloride-injured vena cava in a dose-dependent manner; HAPI M358R was less effective and α1-PI M358R was without effect. In a model of murine endotoxaemia, HAPI RCL5 was more effective than α1-PI M358R in reducing radiolabelled fibrin(ogen) deposition in heart and kidneys; immunohistochemistry of tissue sections showed lesser staining with anti-fibrin(ogen) antibodies with both treatments. In the ferric chloride-injured murine carotid artery, administration of both recombinant serpins was equally effective in lengthening the vessel's time to occlusion. Our results show that the antithrombotic efficacy of the recombinant serpins correlates with their potency as thrombin inhibitors, since HAPI RCL5 inhibits thrombin, but not factors Xa, XIa, XIIa, or neutrophil elastase, more rapidly than α1-PI M358R.

Effectiveness of dabigatran etexilate for thromboprophylaxis of mechanical heart valves.

OBJECTIVE: Warfarin reduces risk of stroke in patients with mechanical heart valves but increases risk of hemorrhage and is difficult to use. Dabigatran etexilate, a new oral direct thrombin inhibitor, is safe and effective in reducing risk of stroke among patients with atrial fibrillation. No data exist in the setting of mechanical heart valves. We tested the hypothesis that dabigatran etexilate is as effective as heparin for thromboprophylaxis of mechanical valves in a porcine heterotopic aortic valve model. METHODS: Thirty swine underwent implantation of modified bileaflet mechanical valved conduit bypassing the ligated, native descending thoracic aorta. Animals randomly received no anticoagulation (n = 10), enoxaparin 2 mg/kg subcutaneously twice daily (n = 10), or dabigatran etexilate 20 mg/kg orally twice daily. Primary end point was amount of valve thrombus at 30 days. Secondary end points included quantitative measurement of platelet deposition on valve prosthesis, thromboelastography, and hemorrhagic and embolic events. RESULTS: At 30 days, we observed 638 ± 895 mg thrombus in no anticoagulation group, 121 ± 128 mg in enoxaparin group, and 19 ± 31 mg in dabigatran etexilate group (P = .01 enoxaparin vs dabigatran etexilate). Fewer platelets were deposited on valves in dabigatran etexilate group (2.7 × 10(8)) than in enoxaparin group (1.8 × 10(9), P = .03). No major or occult hemorrhagic or embolic events were observed. By thromboelastographic analysis, dabigatran etexilate produced less prolongation of K value (P = .01) and less decreases in angle (P = .01) and maximum amplitude (P = .001) than enoxaparin. CONCLUSIONS: Dabigatran etexilate is as effective as enoxaparin for short-term thromboprophylaxis of mechanical valves. It prevents valve thrombus and platelet deposition at 30 days without increased adverse events. These promising results serve as a foundation for prospective clinical trials with dabigatran etexilate as an alternative to warfarin in patients with bileaflet mechanical aortic valves.

Differential effects of TAK-442, a novel orally active direct factor Xa inhibitor, and ximelagatran, a thrombin inhibitor, on factor V-mediated feedback on coagulation cascade and bleeding.

Thrombin amplifies the blood coagulation via factor V (FV)-mediated positive feedback loop. We hypothesised that factor Xa (FXa) inhibitors would interfere more gradually with this feedback activation loop than thrombin inhibitors, thereby achieving a better balance between haemostasis and prevention of thrombosis. In this study, we compared the effects of TAK-442, a novel FXa inhibitor, versus ximelagatran, a thrombin inhibitor, on FV-mediated positive feedback, venous thrombosis and bleeding. In normal plasma, TAK-442 delayed the onset of tissue factor-induced thrombin generation and prolonged prothrombin time (PT) with more gradual concentration-response curve than melagatran, the active form of ximelagatran. The effect of melagatran on the onset of thrombin generation decreased in an FVa-concentration-dependent manner in FV-deficient plasma supplemented with FVa. Furthermore, in FV-deficient plasma, the PT-prolonging potency of melagatran was markedly increased with a change in its concentration-response curve from steep to gradual. In the rat venous thrombosis model, TAK-442 (10 mg/kg, p.o.) prevented thrombus formation by 55% with 1.2 times prolongation of PT; a similar effect was observed in ximelagatran-treated (3 mg/kg, p.o.) rats. TAK-442 at 100 mg/kg prolonged PT by only 2.1 times with no change in bleeding time (BT), whereas ximelagatran at 10 mg/kg prolonged PT by 3.9 times and significantly increased BT. These results suggest that the differential effects of the two agents on FV-mediated amplification of thrombin generation may underlie the observation of a wider therapeutic window for TAK-442 than for ximelagatran.

Evaluation of AR-H067637, the active metabolite of the new direct thrombin inhibitor AZD0837, in models of venous and arterial thrombosis and bleeding in anaesthetised rats.

AZD0837, currently in clinical development, is a once-daily oral anticoagulant that is bioconverted to AR-H067637, a selective, reversible direct thrombin inhibitor (DTI). When developing a new DTI, the antithrombotic effects are commonly investigated in in vivo animal models; this report shows the effect of AR-H067637 in venous and arterial thrombosis and bleeding models in anaesthetised rats. Thrombus formation was induced by topical application of ferric chloride to the carotid artery or to the caval vein with partial stasis. Cutaneous incision bleeding time and muscle transection blood loss were assessed, with or without acetylsalicylic acid (ASA). Activated partial thromboplastin time (APTT), ecarin coagulation time (ECT) and thrombin coagulation time (TCT) were used as plasma biomarkers of anticoagulant effect. Dalteparin was used as a reference compound. AR-H067637, given by continuous infusion, displayed a dose-dependent antithrombotic effect, with 50% inhibition (IC50) of thrombus size in venous and arterial thrombosis models obtained at plasma concentrations of 0.13 µM and 0.55 µM, respectively, without increased bleeding. Dose-dependent increased bleeding and blood loss were seen at plasma concentrations ≥1 µM AR-H067637. At the highest AR-H067637 plasma concentration tested, bleeding time and blood loss increased two and four times the vehicle group. Addition of ASA moderately potentiated bleeding time and blood loss. APTT, ECT and TCT were dose-dependently prolonged. These studies demonstrate that the DTI AR-H067637 inhibits thrombus formation in rat venous and arterial thrombosis models with no or minor increases in bleeding.

Thrombin inhibitors identified by computer-assisted multiparameter design.

Here, we present a series of thrombin inhibitors that were generated by using powerful computer-assisted multiparameter optimization process. The process was organized in design cycles, starting with a set of randomly chosen molecules. Each cycle combined combinatorial synthesis, multiparameter characterization of compounds in a variety of bioassays, and algorithmic processing of the data to devise a set of compounds to be synthesized in the next cycle. The identified lead compounds exhibited thrombin inhibitory constants in the lower nanomolar range. They are by far the most selective synthetic thrombin inhibitors, with selectivities of >100,000-fold toward other proteases such as Factor Xa, Factor XIIa, urokinase, plasmin, and Plasma kallikrein. Furthermore, these compounds exhibit a favorable profile, comprising nontoxicity, high metabolic stability, low serum protein binding, good solubility, high anticoagulant activity, and a slow and exclusively renal elimination from the circulation in a rat model. Finally, x-ray crystallographic analysis of a thrombin-inhibitor complex revealed a binding mode with a neutral moiety in the S1 pocket of thrombin.