Cardiac-Specific Overexpression of Oxytocin Receptor Leads to Cardiomyopathy in Mice.

BACKGROUND: Oxytocin (Oxt) and its receptor (Oxtr) gene system has been implicated in cardiomyogenesis and cardioprotection; however, effects of chronic activation of Oxtr are not known. We generated and investigated transgenic (TG) mice that overexpress Oxtr specifically in the heart. METHODS AND RESULTS: Cardiac-specific overexpression of Oxtr was obtained by having the α-major histocompatibility complex promoter drive the mouse Oxtr gene (α-Mhc-Oxtr). Left ventricular (LV) function and remodeling were assessed by magnetic resonance imaging and echocardiography. In α-Mhc-Oxtr TG mice, LV ejection fraction was severely compromised at 14 weeks of age compared with wild-type (WT) littermates (25 ± 6% vs 63 ± 3%; P < .001). LV end-diastolic volume was larger in the TG mice (103 ± 6 µL vs 67 ± 5 µL; P < .001). α-Mhc-Oxtr TG animals displayed cardiac fibrosis, atrial thrombus, and increased expression of pro-fibrogenic genes. Mortality of α-Mhc-Oxtr TG animals was 45% compared with 0% (P < .0001) of WT littermates by 20 weeks of age. Most cardiomyocytes of α-Mhc-Oxtr TG animals but not WT littermates (68.0 ± 12.1% vs 5.6 ± 2.4%; P = .008) were positive in staining for nuclear factor of activated T cells (NFAT). To study if thrombin inhibitor prevents thrombus formation, a cohort of 7-week-old α-Mhc-Oxtr TG mice were treated for 12 weeks with AZD0837, a potent thrombin inhibitor. Treatment with AZD0837 reduced thrombus formation (P < .05) and tended to attenuate fibrosis and increase survival. CONCLUSIONS: Cardiac-specific overexpression of Oxtr had negative consequences on LV function and survival in mice. The present findings necessitate further studies to investigate potential adverse effects of chronic Oxt administration. We provide a possible mechanism of Oxtr overexpression leading to heart failure by nuclear factor of activated T cell signaling. The recapitulation of human heart failure and the beneficial effects of the antithrombin inhibitor render the α-Mhc-Oxtr TG mice a promising tool in drug discovery for heart failure.

Effects on fibrin network porosity of anticoagulants with different modes of action and reversal by activated coagulation factor concentrate.

Orally available direct thrombin inhibitors (DTI) and direct activated factor X inhibitors (DFXaI) may replace vitamin K antagonists in patients needing long-term anticoagulant treatment. We investigated the influence on the fibrin network of anticoagulants with different modes of action: AR-H067637 (DTI), the active metabolite of AZD0837, apixaban (DFXaI), fondaparinux (indirect FXaI) and warfarin. Counteraction of the anticoagulant effect by FEIBA(®) (Factor Eight Inhibitor Bypass Activity) was also investigated. Tissue factor, phospholipids and calcium were used to initiate coagulation in human platelet poor plasma. The permeability constant (Ks), reflecting the amount of buffer passing through the coagulum, was calculated and the fibrin network was visualized by 3D confocal microscopy. Warfarin (International Normalized Ratio 2-3) increased Ks in plasma by 28-50% compared with control. 'Therapeutic' plasma concentrations of AR-H067637 (0·3-0·6 µmol/l), apixaban (0·2-0·4 µmol/l) and fondaparinux (0·1-0·3 µmol/l) increased Ks by 72-91%, 58-76% and 36-53% respectively. Addition of FEIBA(®) totally reversed the warfarin effect but only partially reversed effects of the other anticoagulants at concentrations that increased Ks by 50% or more. Fibrin network observed with 3D confocal microscopy agreed well with the permeability results. In conclusion, all examined anticoagulants rendered the fibrin network more porous. FEIBA(®) reversed the increased permeability in warfarin plasma but had only partial effects on the other anticoagulants.

Biochemical and pharmacological effects of the direct thrombin inhibitor AR-H067637.

AZD0837 is in development as a new oral anticoagulant for use in thromboembolic disorders. In vivo, AZD0837 is converted to AR-H067637, a selective and reversible direct thrombin inhibitor. Established biochemical methods were used to assess and measure the biochemical and pharmacological properties of AR-H067637. Both direct Biacore binding studies of AR-H067637 with immobilised alpha-thrombin and inhibition studies using pre-steady state kinetics with thrombin in the fluid phase confirmed that AR-H067637 is a rapid-binding, reversible and potent (inhibition constant K(i) = 2-4 nM), competitive inhibitor of thrombin, as well as of thrombin bound to fibrin (clot-bound thrombin) or to thrombomodulin. The total amount of free thrombin generated in platelet-poor clotting plasma was inhibited concentration-dependently by AR-H067637, with a concentration giving half maximal inhibition (IC(50)) of 0.6 microM. Moreover, AR-H067637 is, with the exception of trypsin, a selective inhibitor for thrombin without inhibiting other serine proteases involved in haemostasis. Furthermore, no anticoagulant effect of the prodrug was found. AR-H067637 prolonged the clotting time concentration-dependently in a range of plasma coagulation assays including activated partial thromboplastin time, prothrombin time, prothrombinase-induced clotting time, thrombin time and ecarin clotting time. The two latter assays were found to be most sensitive for assessing the anticoagulant effect of AR-H067637 (plasma IC(50) 93 and 220 nM, respectively). AR-H067637 also inhibited thrombin-induced platelet activation (by glycoprotein IIb/IIIa exposure, IC(50) 8.4 nM) and aggregation (IC(50) 0.9 nM). In conclusion, AR-H067637 is a selective, reversible, competitive inhibitor of alpha-thrombin, with a predictable anticoagulant effect demonstrated in plasma coagulation assays.

The direct thrombin inhibitor melagatran counteracts endotoxin-induced endothelial leukocyte adherence and microvascular leakage in the rat mesentery. Rationale for the treatment of inflammatory disorders beyond sepsis?

The activation of the coagulation system in the course of an inflammatory reaction impairs the function of the microcirculation. By means of intravital videomicroscopy the effect of the direct thrombin inhibitor melagatran on endotoxin-induced microvascular permeability and leukocyte adhesion to microvascular endothelium of rat mesentery was evaluated. Secondly, plasma concentrations of melagatran or interleukin-6 in response to endotoxin or after treatment with melagatran respectively, were determined. Male Sprague-Dawley rats (300-400 g bw) were infused with 0.5 mg/kg lipopolysaccharide (LPS) (E. coli O55:B5) over 80 minutes. Vascular leakage was detected with FITC-marked rat serum albumin by fluorescence microscopy and evaluated by grey value analysis with a computer assisted image processing system. Light microscopy was used to evaluate the adherence of leukocytes to the vessel wall. Two treated groups received either 0.3 or 0.6 mg/kg bw melagatran iv in addition to LPS-infusion. The observation period was 3 hours after the beginning of LPS infusion. Groups of animals not infused with LPS or solely treated with melagatran (0.3 or 0.6 mg/kg) served as controls. Infusion of LPS led to a significant increase of microvascular permeability, leukocyte adherence and thrombin-antithrombin complex plasma concentration compared to unstimulated controls. These effects were significantly reduced by melagatran at both dosage levels. Elevated plasma concentrations of melagatran were observed in animals infused with endotoxin and higher plasma levels of interleukin-6 were found in endotoxemic animals treated with melagatran. The results indicate that thrombin is one of the most important clotting enzymes involved in inflammatory microvascular disturbance. Moreover, it should be clarified whether direct thrombin inhibitors themselves play a role within the immune response to endotoxin.

Antithrombotic effects of ximelagatran plus acetylsalicylic acid (ASA) and clopidogrel plus ASA in a human ex vivo arterial thrombosis model.

It was the objective of this study to compare the antithrombotic effects and bleeding profiles of the oral direct thrombin inhibitor ximelagatran, an anticoagulant, and the antiplatelet agent clopidogrel on top of steady-state acetylsalicylic acid (ASA) in a human arterial thrombosis model. Healthy male volunteers (n=62) received ASA (160 mg once daily), plus either clopidogrel for 6 days (loading dose 300 mg, then 75 mg once daily), or a single dose of ximelagatran (36 or 72 mg) on Day 6. Changes in total thrombus area (TTA) under low shear rate (LSR; 212 s(-1)) and high shear rate (HSR; 1690 s(-1)) conditions were measured, using the ex vivo Badimon perfusion chamber model pre-dose and 2 and 5 hours after dosing on Day 6, and capillary bleeding times (CBT) were determined. Ximelagatran plus ASA significantly reduced TTA under LSR and HSR, compared with ASA alone. Ximelagatran plus ASA reduced TTA more than clopidogrel plus ASA under LSR after 2 hours (36 mg, P=0.0011; 72 mg, P<0.0001) and 5 hours (72 mg, P=0.0057), and under HSR after 2 and 5 hours (72 mg, P<0.05). Compared with ASA alone, CBT was markedly prolonged by clopidogrel plus ASA (ratio 6.4; P<0.0001) but only slightly by ximelagatran plus ASA (72 mg ximelagatran, ratio 1.4; P=0.0010). Both drug combinations were well tolerated. Oral ximelagatran plus ASA has a greater antithrombotic effect in this human ex vivo thrombosis model and a less pronounced prolongation of bleeding time than clopidogrel plus ASA.

The effects of ximelagatran and warfarin on the prophylaxis of a caval vein thrombosis and bleeding in the anaesthetized rat.

The antithrombotic and bleeding properties of the oral direct thrombin inhibitor ximelagatran and of warfarin were investigated in an experimental venous thrombosis and bleeding model in anaesthetized rats. Rats were randomized to receive ximelagatran (1-20 micromol/kg), warfarin (0.20-0.82 micromol/kg), or vehicle (tap water) once daily orally for 4 days before surgery. Thrombosis was induced by partial stenosis and application of ferric chloride to the wall of the abdominal vena cava under anaesthesia. Sixty minutes after thrombus induction, rats were sacrificed, thrombi harvested, and their fresh weight determined. Bleeding was determined as haemoglobin in fluid collected from the abdominal cavity. Blood samples were taken before thrombus induction and sacrifice for determination of coagulation parameters and plasma concentrations of melagatran, the active form of ximelagatran. Ximelagatran and warfarin dose-dependently reduced thrombus formation. The highest doses of ximelagatran and warfarin almost completely prevented thrombus formation; however, the increase in bleeding (versus vehicle) was significantly lower with the highest dose of ximelagatran than with the highest dose of warfarin. The oral direct thrombin inhibitor ximelagatran is thus as at least as effective as warfarin in the prevention of thrombus formation in this animal model, but with a wider separation between antithrombotic effects and bleeding.

Effect of recombinant factor VIIa on melagatran-induced inhibition of thrombin generation and platelet activation in healthy volunteers.

The objectives were to investigate whether activation of the extrinsic coagulation cascade by recombinant factor VIIa (rFVIIa) reverses the inhibition of thrombin generation and platelet activation by melagatran, the active form of the oral direct thrombin inhibitor ximelagatran. In a single-blind, randomized, parallel-group study, volunteers (20 per group) received a 5-hour intravenous (i.v.) infusion to achieve steady-state melagatran plasma concentrations of approximately 0.5 micromol/L, with a single i.v. bolus of rFVIIa (90 microg/kg) or placebo at 60 minutes. Prothrombin fragment 1+2, thrombin-anti-thrombin complex, fibrinopeptide A, beta-thromboglobulin, and thrombin-activatable fibrinolysis inhibitor were quantified for venous and shed blood. Activated partial thromboplastin time (APTT), prothrombin time (PT), endogenous thrombin potential, thrombus precursor protein (TpP), and plasmin-alpha(2)-antiplasmin complex concentrations were determined in venous blood. Shed blood volume was measured. Melagatran reduced markers of thrombin generation and platelet activation in shed blood and prolonged APTT. rFVIIa increased FVIIa activity, PT, and TpP in venous blood. All other parameters were unaffected. In conclusion, rFVIIa did not reverse the anticoagulant effects of high constant concentrations of melagatran. However, the potential value of higher, continuous or repeated doses of rFVIIa or its use with lower melagatran concentrations has not been excluded.

The pharmacodynamics and pharmacokinetics of the oral direct thrombin inhibitor ximelagatran and its active metabolite melagatran: a mini-review.

Ximelagatran (Exanta, AstraZeneca) is a novel, oral direct thrombin inhibitor (oral DTI) that is rapidly converted to melagatran, its active form, following absorption. Melagatran has been shown to be a potent, rapidly binding, competitive inhibitor of human alpha-thrombin that inhibits both thrombin activity and generation. Melagatran also effectively inhibits both free and clot-bound thrombin. Melagatran has a wide therapeutic interval that enables it to be administered safely across a wide range of doses with no increased risk of bleeding, in contrast with warfarin whose narrow therapeutic window necessitates monitoring of its pharmacodynamic effect. Although melagatran has all the pharmacodynamic properties required of a new antithrombotic agent, low oral bioavailability that is even further reduced by the concomitant intake of food precludes its development as an oral agent. It was this that propelled the development of its prodrug, ximelagatran, which is 170 times more lipophilic than melagatran and uncharged at intestinal pH. Ximelagatran is therefore much better than melagatran at penetrating the gastrointestinal barrier and, as a consequence, has sufficient bioavailability (20%) for oral administration. Moreover, its pharmacokinetic properties following oral administration are stable and reproducible, with no food interactions and a low potential for drug-drug interactions. These properties allow ximelagatran to be administered twice daily according to a fixed dose regimen without coagulation monitoring. As a consequence of its favourable pharmacokinetic and pharmacodynamic properties, ximelagatran is currently undergoing full-scale clinical development for the prophylaxis and treatment of thromboembolic disorders.

Effects of ximelagatran, the oral form of melagatran, in the treatment of caval vein thrombosis in conscious rats.

The antithrombotic effects of direct (ximelagatran and hirudin) and indirect (dalteparin) anticoagulants were compared using a deep venous thrombosis (DVT) treatment model in conscious rats. Thrombus formation was induced in the inferior caval vein by total stasis plus topically applied ferric chloride. After 1-h thrombus maturation, one group of 10 rats were sacrificed and the mean thrombus weight in this group was 27.3 +/- 2.7 mg. This thrombus weight was handled as a reference to which all other results were compared. In all other groups, the total occlusion was removed after 1 h but a partial stasis was retained, permitting some blood flow around the thrombus. Groups of animals received subcutaneous (s.c.) dalteparin (200 IU/kg), s.c. hirudin (0.75 micromol/kg), one of four oral doses of ximelagatran (2.5, 5, 10 or 20 micromol/kg) or s.c. saline (control). After the 3-h treatment, mean thrombus weight in the saline group (26.5 +/- 3.3 mg) did not differ significantly from that of the reference group (27.3 +/- 2.7 mg, see above). Ximelagatran decreased thrombus weight in a dose-dependent manner, with an estimated ID(50) of 15 micromol/kg. Mean thrombus weight with the highest ximelagatran dose (11.1 +/- 1.3 mg) was similar to that with hirudin (13.0 +/- 1.5 mg). The effect of dalteparin on thrombus regression was much less pronounced (20.2 +/- 1.2 mg), compared with ximelagatran and hirudin, even though it was administered at a dose that yielded a similar activated partial thromboplastin time (APTT) prolongation. In conclusion, the results from this DVT treatment model showed that direct thrombin inhibitors ximelagatran and hirudin exhibited superior antithrombotic properties to low molecular weight heparin (LMWH).

Expression and characterization of recombinant ecarin.

The snake venom protease ecarin from Echis carinatus was expressed in stable transfected CHO-S cells grown in animal component free cell culture medium. Recombinant ecarin (r-ecarin) was secreted from the suspension adapted Chinese Hamster Ovary (CHO-S) host cells as a pro-protein and activation to the mature form of r-ecarin occurred spontaneously during continued incubation of the cell culture at 37 °C after death of the host cells. Maximal ecarin activity was reached 7 days or more after cell culture viability had dropped to zero. The best producing CHO-S clone obtained produced up to 7,000 EU ecarin/litre in lab scale shaker cultures. The conversion of different concentrations of both prothrombin and prethrombin-2 as substrates for native and r-ecarin were examined with a chromogenic thrombin substrate. At low concentrations both these proteins were converted into thrombin by the two ecarin preparations with comparable rates. However, with prothrombin concentrations above 250 nM r-ecarin apparently had a two times higher turnover than native ecarin, consistent with the observed rapid complete conversion of prothrombin into thrombin by r-ecarin. With r-ecarin a K (m) value of 0.4 µM prethrombin-2 was determined but only a rough estimate could be made of the K (m) for prothrombin of 0.9 µM. In conclusion, r-ecarin was identified as a promising candidate for replacement of native ecarin in assays utilizing conversion of prothrombin to thrombin.

Effect on perfusion chamber thrombus size in patients with atrial fibrillation during anticoagulant treatment with oral direct thrombin inhibitors, AZD0837 or ximelagatran, or with vitamin K antagonists.

INTRODUCTION: AZD0837 and ximelagatran are oral direct thrombin inhibitors that are rapidly absorbed and bioconverted to their active forms, AR-H067637 and melagatran, respectively. This study investigated the antithrombotic effect of AZD0837, compared to ximelagatran and the vitamin K antagonist (VKA) phenprocoumon (Marcoumar), in a disease model of thrombosis in patients with non-valvular atrial fibrillation (NVAF). METHODS: Open, parallel-group studies were performed in NVAF patients treated with VKA, which was stopped aiming for an international normalized ratio (INR) of ≤ 2 before randomization. Study I: 38 patients randomized to AZD0837 (150,250 or 350 mg) or ximelagatran 36 mg twice daily for 10-14 days. Study II: 27 patients randomized to AZD0837 250 mg twice daily or VKA titrated to an INR of 2-3 for 10-14 days. A control group of 20 healthy elderly subjects without NVAF or anticoagulant treatment was also studied. Size of thrombus formed on pig aorta strips was measured after a 5-minute perfusion at low shear rate with blood from the patient/control subject. RESULTS: Thrombus formation was inhibited by AZD0837 and ximelagatran. Relative to untreated patients, a 50% reduction of thrombus size was estimated at plasma concentrations of 0.6 and 0.2 µmol/L for AR-H067637 and melagatran, respectively. For patients receiving VKA treatment, the thrombus size was about 15% lower compared with healthy elderly controls. CONCLUSIONS: Effects of AZD0837 and ximelagatran on thrombus formation were similar or greater than for VKA therapy and correlated with plasma concentrations of their active forms.

Both ADP and thrombin regulate arteriolar thrombus stabilization and embolization, but are not involved in initial hemostasis as induced by micropuncture.

OBJECTIVE: Thrombosis and embolization are main causes of morbidity and mortality. Up to now, the relative importance of mediators involved is only partly known. It was the aim of this study to investigate the involvement of ADP and thrombin in subsequent phases of arteriolar hemostasis and thromboembolism in vivo. METHODS: Rabbit mesenteric arterioles were punctured, which induced bleeding, hemostasis, and subsequent thromboembolism. This reaction as well as the activation state of platelets involved ([Ca(2+)](i)), was monitored in real time by intravital (fluorescence) microscopy. RESULTS: Neither inhibition of thrombin formation or thrombin activity nor blockade of platelet ADP receptors P2Y(1) and P2Y(12) influenced the initial hemostatic reaction: in all experiments initial bleeding was stopped by a primary thrombus within 2-3 s. On the other hand, both thrombin inhibition and P2Y(1) blockade increased rebleeding frequency, which indicates reduced thrombus stability in the long term. Finally, inhibition of either thrombin or ADP (via both receptors) reduced aggregate formation during the embolization phase by at least 90%. While most participating platelets exhibited a transient increase in [Ca(2+)](i) during embolization, an increased percentage of platelets showed no calcium response at all during P2Y(1) blockade, which was accompanied by reduced platelet-platelet interaction strength. CONCLUSIONS: Whereas thrombin and ADP are not involved in the initial hemostatic reaction, both substances appear to be essential to prevent rebleedings in the long term. During subsequent embolization, ADP (via both receptors) and small amounts of thrombin are involved in platelet activation.

A combination of a thrombin inhibitor and dexamethasone prevents the development of experimental disseminated intravascular coagulation in rats.

INTRODUCTION: Disseminated intravascular coagulation (DIC) is a serious and potentially lethal complication of severe sepsis. DIC is characterised primarily by widespread platelet aggregation and fibrin deposition, followed by consumption of platelets, coagulation factors, and inhibitors. The aim of the study was to evaluate the efficacy of the active-site thrombin inhibitor melagatran, the active form of the oral direct thrombin inhibitor ximelagatran, in reducing fibrinogen and platelet consumption in blood and fibrin deposition in organs, in an experimental endotoxinaemia rat model. MATERIALS AND METHODS: In this model, DIC was induced by an intravenous injection of endotoxin (1 mg/kg). Melagatran was compared with unfractionated heparin and the synthetic glucocorticoid analogue dexamethasone. Animals were divided into 16 treatment groups in which high and low doses of each agent were tested alone and in combination with melagatran. RESULTS: Fibrinogen consumption was reduced by melagatran, dexamethasone, and heparin, and was completely prevented by melagatran in combination with dexamethasone. Platelet consumption was partially reduced by melagatran, unfractionated heparin, and dexamethasone, but complete protection was observed only with melagatran in combination with dexamethasone. Melagatran in combination with dexamethasone or heparin protected the liver and spleen from fibrin deposition. CONCLUSION: In this experimental DIC rat model, the direct thrombin inhibitor melagatran given together with dexamethasone protected against the consequences of activated haemostasis.

Three vehicle formulations for melagatran, a direct thrombin inhibitor, evaluated in a vena cava thrombosis model in the rat.

BACKGROUND: The objective of this study was to investigate whether the use of a depot formulation would enhance the antithrombotic effect of the direct thrombin inhibitor melagatran. METHODS AND RESULTS: In a rat venous thrombosis model, animals were openly randomized to receive subcutaneously (s.c.) either vehicle (saline, cyclodextrin or poloxamer) or melagatran (0.5 microM/kg) dissolved in vehicle. An additional injection of cyclodextrin or poloxamer was given at another site to investigate whether the vehicle itself had any additional effect. All injections were given 30 min before induction of thrombus formation. Thrombus formation was induced by ferric chloride, together with stenosis of the caval vein, during a short period of inhalation anaesthesia. Five hours later the thrombi were harvested and their wet weight determined. Thrombus size was comparable across the vehicle-only groups. The antithrombotic effects of melagatran in saline or poloxamer were comparable while melagatran in cyclodextrin was less effective. The effects of melagatran in saline, cyclodextrin or poloxamer were not enhanced by additional cyclodextrin or poloxamer. Thrombin time (TT) and activated partial thromboplastin time (aPTT) at the end of the experiment were prolonged to a greater extent in the groups receiving melagatran in cyclodextrin or poloxamer compared with those receiving melagatran in saline. CONCLUSION: In this vena cava thrombosis model, no enhanced antithrombotic effect was observed with melagatran given as a s.c. depot formulation in cyclodextrin or poloxamer compared with that in saline.