Thrombin Inhibitor Melagatran Research Articles

Ximelagatran for the Prevention of Venous Thromboembolism Following Elective Hip or Knee Replacement Surgery

Patients undergoing major lower-extremity orthopedic surgery such as total hip replacement (THR) and total knee replacement (TKR) are at an increased risk of venous thromboembolism (VTE). Routine prophylaxis is necessary to reduce the risk of deep vein thrombosis (DVT), which may progress to potentially fatal pulmonary embolism and secondary complications such as postthrombotic syndrome, recurrent DVT, and chronic pulmonary hypertension. Prophylaxis in patients undergoing TKR, THR, and hip fracture surgery is now standard practice and generally involves anticoagulant treatment with either low-molecular-weight heparin (LMWH) or warfarin for a period of 7 to 10 days, with extended prophylaxis in those with ongoing risk factors such as obesity, cancer, or previous VTE. Data from clinical practice suggest that there is a general trend toward longer postsurgical prophylaxis and shorter hospital stays, making practicality of treatment an important consideration. LMWH is effective for the prophylaxis of VTE, but the parenteral route of administration is not convenient for use in the outpatient setting. Warfarin, on the other hand, can be administered orally but requires the infrastructure for careful patient monitoring and dose adjustments because of its unpredictable dose-response relationship. The development of new anticoagulants has been pursued with the aim of improving efficacy, predictability, consistency of response, safety, and convenience. A recently approved anticoagulant, fondaparinux, has been proven to provide superior efficacy for the prevention of VTE compared with LMWH, but this agent requires parenteral administration and does not overcome the convenience issue. Ximelagatran is the oral form of the direct thrombin inhibitor melagatran, which is available for subcutaneous administration. Ximelagatran has a consistent anticoagulant response allowing fixed oral dosing without the need for coagulation monitoring. The efficacy and safety profile of melagatran/ximelagatran prophylaxis for VTE following THR and TKR has compared favorably with standard LMWH prophylaxis, as seen in the European METHRO II and III trials and EXPRESS trial, and with warfarin prophylaxis, as seen in the North American EXULT A and B trials. Several prophylactic treatment regimens have been evaluated in the European trials to determine the optimal dosing and timing of first dose of melagatran to achieve the best balance of efficacy and safety. Preoperative initiation of melagatran was more effective than when prophylactic treatment was initiated postoperatively, and the lowest rates of bleeding were associated with a postoperative initiation of prophylaxis. Early administration of the first postoperative melagatran dose (4 to 8 hours) was also associated with better prophylactic efficacy relative to a later postoperative start (8 to 12 hours). The results of the comprehensive international clinical trial program and in particular the optimal balance of efficacy/safety data provided by the METHRO III study have led to approval of melagatran/ximelagatran in 2004 in the European Union for the prevention of VTE in patients undergoing elective hip or knee replacement surgery. Ximelagatran has the potential to maximize the use of anticoagulation in patients discharged following major lower-extremity orthopedic surgery.

Prevention of clot formation during haemodialysis using the direct thrombin inhibitor melagatran in patients with chronic uraemia

This study assessed the feasibility of replacing intravenous (i.v.) dalteparin with the direct thrombin inhibitor (DTI) melagatran administered via dialysis fluid in patients undergoing haemodialysis, and also examined the pharmacokinetics of melagatran with and without dialysis. During two 4 h haemodialysis sessions, 10 adult patients were administered i.v. dalteparin. During two subsequent sessions, melagatran was administered as an i.v. bolus before dialysis, and in the dialysis fluid. The pharmacokinetics of melagatran administered as a bolus before dialysis, and of i.v. melagatran during a dialysis-free day, were studied. Dialysis performances were evaluated from clinical criteria including clot formation in the dialyzer and bloodlines, pre-post dialyzer pressures and iohexol clearance. Anticoagulant efficacy was evaluated from dialysis success. All dialysis sessions were successful, with no apparent difference in clot formation between the two treatments. Median iohexol clearance was similar with dalteparin (99-103 ml/min) and melagatran in the dialysis fluid (98-100 ml/min). There was no difference in pre- and post-dialyzer bloodline pressures between the two treatments. During dialysis sessions with melagatran in dialysis fluid, melagatran concentrations in plasma rapidly equilibrated to approximately 70% of those in dialysis fluid. While the clearance of melagatran was low in patients with renal failure (mean+/-SD, 0.93+/-0.36 l/h), haemodialysis provided efficient clearance of melagatran (7.20+/-0.76 l/h). Melagatran clearance by dialysis (104+/-10 ml/min) was comparable to iohexol clearance. The DTI melagatran administered via dialysis fluid may provide sufficient anticoagulation for haemodialysis. Melagatran is rapidly cleared from plasma by haemodialysis, suggesting that this method may be used to decrease drug levels in patients with renal impairment.

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.

The Role of the Innate Immunity in Islet Transplantation

Clinical islet transplantation is an emerging procedure to cure type 1 diabetes. The graft is implanted by infusion into the liver through the portal vein. A major obstacle that still needs to be overcome is the requirement for islets from multiple donors to achieve insulin independence.An innate inflammatory reaction, the IBMIR, is elicited when islets are exposed to blood. The IBMIR has been described as a clotting reaction culminating in disruption of islet morphology and is a plausible cause for loss of tissue during the early post-transplant period. In this thesis, the underlying mechanisms of the IBMIR were characterized. The IBMIR was for the first time demonstrated in patients undergoing an islet transplant, and a number of clinically applicable strategies to limit this reaction were identified.The thrombin inhibitor melagatran completely blocked the IBMIR in an in vitro tubing blood loop system, indicating that thrombin is the driving force in the reaction. Interestingly, islets were shown to produce and secrete tissue factor (TF), the physiological trigger of coagulation. Inactivated FVIIa, a specific inhibitor of TF, successfully blocked initiation of the IBMIR. An alternative approach to limit the IBMIR was to pre-treat islets in culture prior to transplantation. Nicotinamide added to the culture medium effectively decreased the level of TF in human islets. Infiltration of immune cells, also a part of the IBMIR, was characterized in detail. The predominant cell types infiltrating the islets were neutrophilic granulocytes and, to a lesser degree, monocytes. Both cell types may exert direct cytotoxic effects, and the antigen-presenting monocytes may also be important for directing the specific immune system to the site of inflammation. These findings have provided new insight into the nature of the IBMIR and offer several new strategies to improve the outcome of clinical islet transplantation.

Advances in Anticoagulation Therapy: The Role of Selective Inhibitors of Factor Xa and Thrombin in Thromboprophylaxis after Major Orthopedic Surgery

A new generation of antithrombotic agents that target a single enzyme within the procoagulant cascade is making its way into mainstream clinical practice. Borrowing selectively from the properties of their parent anticoagulant, unfractionated heparin, as well as from those of peptide anticoagulants from reptile or insect venoms, designers of the new drugs have created targeted inhibitors of thrombin, factor Xa, or other specific factors in the procoagulant pathways. These new agents promise efficacy and safety profiles far more favorable than those of conventional anticoagulants for thromboprophylaxis after major orthopedic surgery and require no laboratory monitoring of drug efficacy in this setting. Ximelagatran, the oral "prodrug" of the direct thrombin inhibitor melagatran, is in phase III of clinical development. Clinical trials using various dosages of ximelagatran, sometimes preceded by subcutaneous melagatran, as thromboprophylaxis after total hip or knee replacement surgery have suggested efficacy equal to or better than that of warfarin and enoxaparin. The best dosing regimen and optimal timing of first dose for melagatran and ximelagatran remain to be determined, as do the mechanism and impact of drug disturbance of hepatic function. Fondaparinux, a selective, synthetic inhibitor of factor Xa, has been shown in large clinical trials to be superior to low-molecular-weight heparins and is approved as a fixed once-daily subcutaneous 2.5-mg dose for thromboprophylaxis for hip or knee replacement surgery and after hip fracture repair. Fixed-dose fondaparinux 2.5 mg initiated 6 to 8 hours after surgery achieved superior efficacy and comparable safety in head-to-head comparisons with enoxaparin for the prevention of venous thromboembolism after major orthopedic surgery. Fondaparinux is the only agent approved for use in hip fracture patients in the United States at this time and has recently gained approval for extended prophylaxis in this patient population.

Melagatran and Dynamic Whole Blood Clot Formation - Haemostatic Response to Ex Vivo Recombinant Factor Viia and Activated Prothrombin Complex Concentrate.

The direct thrombin inhibitor melagatran (Exanta™, Astra Zeneca, Sweden) has proven efficient for the prevention and treatment of thromboembolism. Major bleeding complications are rare and may often be managed by discontinuation of the drug; however, in some cases of acute serious bleeding, an effective and instant haemostatic intervention may be needed. Potential haemostatic agents may include recombinant factor VIIa (rFVIIa - NovoSeven®, Novo Nordisk, Denmark) or activated prothrombin complex concentrate (APCC - Feiba®, Baxter, Austria). We hypothesized that melagatran induces abnormal whole blood (WB) clotting profiles and rFVIIa as well as APCC may improve the deteriorated clotting profiles. This study aimed to investigate the effect of ex vivo addition of melagatran to WB from healthy males and explore the haemostatic potential of rFVIIa and APCC. Following informed consent, 15 healthy males with an average age of 34 years were enrolled for blood sampling. Continuous WB coagulation profiles were recorded by ROTEG® thrombelastography employing activation with minute amounts of tissue factor (Innovin® final dilution 1:17,000 ~0.35pM). The initiation phase of WB clot formation was defined by the clotting time (CT - sec). Coagulation raw data were processed to provide dynamic parameters that concur with the propagation of WB coagulation such as maximum velocity (MaxVel - mm*100/sec) and time to maximum velocity (t, MaxVel - sec). Titration experiments (n=10) with ex vivo addition of melagatran to WB corresponding plasma concentrations ranging from 0 to 5.0 μM (12 steps) showed a significant and dose dependent prolongation of the CT and t, MaxVel. The MaxVel of WB clot formation was initially reduced from average 13.8 mm*100/sec (12.2-15.4, 95 % CI) to a plateau level of average 9.6 (7.5–12.2) at concentrations of melagatran ranging from 0.125 μM to 0.50 μM. A further and progressive decline in MaxVel was observed at concentrations of melagatran exceeding 1.0μM. Intervention studies (n=10) were performed ex vivo on WB spiked with melagatran at 0.25, 0.50, 1.0, and 2.0 μM followed by ex vivo addition of rFVIIa at concentrations of 25, 50, 100, and 200 nM or APCC at concentration of 0.5, 1.0, 2.0, and 4.0 U/mL. In all tested concentrations of melagatran, rFVIIa significantly shortened the CT and t, MaxVel, while the reduced MaxVel was not accelerated. No dose-response effect of rFVIIa was detected. In contrast, at all concentration of melagatran, APCC significantly and dose dependently shortened the CT, the t, MaxVel as well as increased the MaxVel. As compared to rFVIIa, the effect of APCC was statistically more potent. At melagatran 0.25 μM, APCC at 1.0, 2.0, and 4.0 U/mL normalized the MaxVel. In all other experimental settings, rFVIIa or APCC did not normalize the dynamic WB coagulation parameters following anticoagulation with melagatran. In conclusion, melagatran induces unique changes of dynamic WB clot formation as illustrated by the prolonged initiation and plateau interval of MaxVel in clot propagation. rFVIIa as well as APCC significantly improved the WB clot formation, although reversal of melagatran anticoagulation was not obtained. The more pronounced effect of APCC may be caused by addition of prothrombin and activated coagulation factors. However, this intervention may be less safe than use of rFVIIa.

The Role of Oral Direct Thrombin Inhibitors in the Prophylaxis of Venous Thromboembolism

Venous thromboembolism, which is manifested as deep vein thrombosis (DVT) and pulmonary embolism (PE), represents a significant cause of death, disability, and discomfort. Two million people/year are affected by VTE, making it the third most common cardiovascular disease after coronary heart disease and stroke. The rationale for VTE prophylaxis stems from the clinically silent presentation of the disease and its prevalence among hospitalized patients. At greatest risk are patients undergoing major orthopedic surgery and those admitted to the intensive care unit with acute myocardial infarction, heart failure, ischemic stroke, respiratory disease, systemic infection, or other medical conditions that immobilize patients for 5 days or longer. Several anticoagulant regimens have been effective in reducing the risk of VTE after major orthopedic surgery. For patients undergoing total hip or knee replacement, treatment with adjusted-dose warfarin, low-molecular-weight heparins, or fondaparinux may be used. Warfarin, which has been around for more than 50 years, is the only oral anticoagulant available for VTE prophylaxis. Ximelagatran, a new low-molecular-weight oral prodrug of the direct thrombin inhibitor melagatran, has advantages over warfarin that may make it the drug of choice for prevention of VTE.

Drotrecogin Alfa (activated, Xigris�) in Combination with Heparin or Melagatran: An In vitro Investigation

The present in vitro study of human plasma investigated the anticoagulant effects of recombinant human activated protein C (rhAPC; drotrecogin alfa [activated, Xigris]), combined with either unfractionated heparin (UH) or the direct thrombin inhibitor melagatran. Prolongation of clotting time and generation of prothrombin fragments 1 and 2 (F1 + 2) and of thrombin-antithrombin (TAT) complex were measured in vitro. Clot formation was induced by adding low levels (final concentration, 20 pmol/L) of lipidated tissue factor (TF) to citrated venous plasma samples from healthy human volunteers (n = 16). It has been suggested that experimental activation of plasma with low levels of TF more closely simulates conditions in vivo. rhAPC, melagatran, or UH alone concentration-dependently prolonged the clotting time and suppressed F1 + 2 and TAT generation. rhAPC-mediated prolongation of clotting time concentration-dependently increased with the addition of either UH or melagatran, the effect being more pronounced with the addition of melagatran. Similarly, rhAPC-mediated generation of F1 + 2 and TAT also increased concentration-dependently with the addition of either UH or melagatran; the effect being, in this case, more pronounced with the addition of UH. This study demonstrated the effects of rhAPC, alone and combined with either UH or melagatran, on clotting time and markers of thrombin generation in human plasma. These results may guide facilitate estimation of appropriate doses of rhAPC and melagatran in future clinical trials.

Thrombin inhibitor reduces myocardial infarction in apoE-/- x LDLR-/- mice.

We have previously shown that atherosclerotic apolipoprotein E-deficient (apoE(-/-)) x LDL receptor-deficient (LDLR(-/-)) mice develop myocardial infarction when exposed to hypoxic stress. This study was performed to assess the role of thrombin and thrombosis in this process. ApoE(-/-) x LDLR(-/-) mice were fed a cholesterol-rich diet for 8 mo and were then subjected to hypoxic stress while receiving isoflurane anesthesia. One group received a bolus dose (5.6 micromol/kg) of the thrombin inhibitor melagatran, and control animals received PBS 10 min before the hypoxic stress. The mice were exposed to 10 min of hypoxia followed by normoxia. Ten minutes after the stress, Alzet pumps delivering melagatran (20 nmol x kg x (-1)min(-1)) or PBS were implanted, and the mice were allowed to recover for 48 h. The cardiac response was analyzed by histology, immunohistochemistry, and serum troponin T assay. All animals showed reversible ECG changes as a sign of ischemia during hypoxic stress, and 50% developed infarctions afterward as judged by troponin T levels. The group that received thrombin inhibitor had significantly lower troponin T and smaller myocardial infarctions than the PBS-treated group. These data show that thrombin generation is an important pathogenetic factor and suggest that coronary thrombosis is involved in myocardial infarction in atherosclerotic mice. Exposure of atherosclerotic mice to hypoxia leads to myocardial infarction through a two-phase pathway in which acute transient ischemia is followed by thrombin-dependent, irreversible, myocardial ischemia and myocardial cell death.

The direct thrombin inhibitor melagatran followed by oral ximelagatran compared with enoxaparin for the prevention of venous thromboembolism after total hip or knee replacement: the EXPRESS study

Ximelagatran and its subcutaneous (s.c.) form melagatran are novel direct thrombin inhibitors for the prevention and treatment of thromboembolic disease. In a double-blind study, 2835 consecutive patients undergoing total hip or knee replacement were randomized to either melagatran/ximelagatran or enoxaparin. Melagatran 2 mg was started immediately before surgery; 3 mg was then administered postoperatively, followed by 24 mg of oral ximelagatran b.i.d. beginning the next day. Enoxaparin 40 mg, administered subcutaneously o.d., was started 12 h before surgery. Both treatments were continued for 8-11 days. The main efficacy outcome measures were major venous thromboembolism (VTE); [proximal deep vein thrombosis (DVT), non-fatal and/or fatal pulmonary embolism (PE), death where PE could not be ruled out], and total VTE (proximal and distal DVT; PE; death from all causes). DVT was detected by mandatory bilateral ascending venography at the end of the treatment period or earlier if clinically suspected. The main safety outcome was bleeding. The rates of major and total VTE were significantly lower in the melagatran/ximelagatran group compared with the enoxaparin group (2.3% vs. 6.3%, P = 0.0000018; and 20.3% vs. 26.6%, P < 0.0004, respectively). Fatal bleeding, critical site bleeding and bleeding requiring reoperation did not differ between the two groups. 'Excessive bleeding as judged by the investigator' was more frequent with melagatran/ximelagatran than with enoxaparin. In patients undergoing total hip or knee replacement, preoperatively initiated s.c. melagatran followed by oral ximelagatran was significantly more effective in preventing VTE than preoperatively initiated s.c. enoxaparin.

Chromogenic substrate as antidote against the thrombin inhibitor 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.

Chromogenes Substrat als Antidot gegen den Thrombininhibitor Melagatran?

ZusammenfassungMit In-vitro-Untersuchungen wird gezeigt, dass ein chromogenes Substrat (Chromozym TH von Roche, Mannheim) die gerinnungshemmende Wirkung des neuen Thrombininhibitors Melagatran (AstraZeneca, Mölndal, Schweden) zumindest partiell antagonisieren kann. Der hier beschriebene Antidoteffekt eines chromogenen Substrats gegenüber einem Thrombininhibitor könnte seine Erklärung darin finden, dass eine Substratkompetition zwischen Thrombins im Testsystem und Fibrinogen, Thrombininhibitor und chromogenem Substrat vorliegt. Tierversuche unter Einbeziehung weiterer Thrombininhibitoren könnten klären, ob die beschriebene Antidotwirkung die beobachteten Blutungskomplikationen tatsächlich reduzieren kann oder ob ein In-vitro-Phänomen vorliegt, das in vivo wenig Relevanz besitzt.

Direct thrombin inhibitor melagatran followed by oral ximelagatran in comparison with enoxaparin for prevention of venous thromboembolism after total hip or knee replacement

We evaluated whether a postoperative regimen with melagatran followed by oral ximelagatran, two new direct thrombin inhibitors, was an optimal regimen for thromboprophylaxis in major orthopaedic surgery. In a double-blind study, 2788 patients undergoing total hip or knee replacement were randomly assigned to receive for 8 to 11 days either 3 mg of subcutaneous melagatran started 4-12 h postoperatively, followed by 24 mg of oral ximelagatran twice-daily or 40 mg of subcutaneous enoxaparin once-daily, started 12 h preoperatively. Ximelagatran was to be initiated within the first two postoperative days. The primary efficacy endpoint was venous thromboembolism (deep-vein thrombosis detected by mandatory venography, pulmonary embolism or unexplained death). The main safety endpoint was bleeding. Venous thromboembolism occurred in 355/1146 (31.0%) and 306/1122 (27.3%) patients in the ximelagatran and enoxaparin group, respectively, a difference in risk of 3.7% in favour of enoxaparin (p = 0.053). Bleeding was comparable between the two groups.

Development and validation of a chiral capillary electrophoresis method for melagatran and ximelagatran drug substances

This work presents the development and validation of an enantioselective capillary electrophoresis (CE) method for two new drug substances, the novel direct thrombin inhibitor melagatran and its oral prodrug ximelagatran. CE with a derivatised cyclodextrin as the chiral discriminant was examined as a viable alternative after failing to achieve sufficient selectivity and sensitivity using chiral liquid chromatography (LC). A robust and sensitive chiral method was developed which utilised a low pH phosphate buffer with heptakis(2,6-Di-O-methyl)-β-cyclodextrin to achieve chiral recognition for both drugs with additional organic modifier to fine tune selectivity. Quantitation at the 0.05% (w/w) level was achieved by employing sample stacking effects possible with a 200 mM phosphate buffer in combination with a narrow 50 μm ID capillary. A full validation was subsequently carried out according to the ICH guidelines, including selectivity, linearity, detection and quantitation limits, accuracy, precision, stability of analytical solutions, and robustness. Linearity, precision, and accuracy data were shown to be adequate over the entire range including at the 0.05% (w/w) quantitation limit. Several electrolyte parameters in addition to buffer depletion and cyclodextrin batch variations were amongst the parameters verified for method robustness.

Inhibition of Thrombin Generation by the Oral Direct Thrombin Inhibitor Ximelagatran in Shed Blood from Healthy Male Subjects

Ximelagatran, an oral direct thrombin inhibitor, whose active form is melagatran, was studied using a model of thrombin generation in humans. Healthy male volunteers (18 per group) received ximelagatran (60 mg p.o.), dalteparin (120 IU/kg s.c.) or a control (water p.o.). Shed blood, collected after incision of the forearm with standardised bleeding time devices at pre-dose, and at 2, 4 and 10 h post-dosing, was analysed for markers of thrombin generation. Statistically significant reductions (p < 0.05) in levels of prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin complex (TAT) in shed blood were detected at 2 and 4 h post-dosing in both the ximelagatran and dalteparin groups. Shed blood F1+2 and TAT levels had returned to pre-dose levels at 10 h post-dosing. Using a shed blood model, we demonstrate that the reversible thrombin inhibitor melagatran and, therefore, oral administration of ximelagatran, inhibits thrombin generation in humans after acute activation of coagulation.

The Direct Thrombin Inhibitor Melagatran and Its Oral Prodrug H 376/95: Intestinal Absorption Properties, Biochemical and Pharmacodynamic Effects

Suboptimal gastrointestinal absorption is a problem for many direct thrombin inhibitors. The studies presented herein describe the new oral direct thrombin inhibitor H 376/95, a prodrug with two protecting residues added to the direct thrombin inhibitor melagatran. Absorption properties in vitro: H 376/95 is uncharged at intestinal pH while melagatran is charged. H 376/95 is 170 times more lipophilic (octanol water partition coefficient) than melagatran. As a result, the permeability coefficient across cultured epithelial Caco-2 cells is 80 times higher for H 376/95 than for melagtran. Pharmacokinetic studies in healthy volunteers: H 376/95 is converted to melagatran in man. Oral bioavailability, measured as melagatran in plasma, is about 20% after oral administration of H 376/95, which is 2.7–5.5 times higher than after oral administration of melagatran. The variability in the area under the drug plasma concentration vs. time curve (AUC) is much smaller with oral H 376/95 (coefficient of variation 20%) than with oral melagatran (coefficient of variation 38%). Pharmacodynamic properties: H 376/95 is inactive towards human α-thrombin compared with melagatran [inhibition constant ( K i) ratio, 185 times], a potential advantage for patients with silent gastrointestinal bleeding. In an experimental thrombosis model in the rat, oral H 376/95 was more effective than the subcutaneous low molecular weight heparin dalteparin in preventing thrombosis. Conclusion: By the use of the prodrug principle, H 376/95 endows the direct thrombin inhibitor melagatran with pharmacokinetic properties required for oral administration without compromising the promising pharmacodynamic properties of melagatran.

Transient isotachophoresis for sensitivity enhancement in capillary electrophoresis-mass spectrometry for peptide analysis.

Transient isotachophoresic (ITP) focusing was used for the on-line analysis of peptides by capillary zone electrophoresis-mass spectrometry (CZE-MS), allowing injection volumes of up to 0.9 microL. A sheath liquid electrospray interface was used with a single quadrupole mass analyzer. First, the technique was applied to the qualitative analysis of a tryptic digest of cytochrome c, resulting in low-background, high-quality spectra. Second, the linear range was investigated by selected ion monitoring (SIM) for a peptidomimetic direct thrombin inhibitor melagatran (Mr 429.5) and two endogenous peptides, substance P (Mr 1348) and calcitonin gene-related peptide (alpha-CGRP; Mr 3806).

Antithrombotic Effects and Bleeding Time of Thrombin Inhibitors and Warfarin in the Rat

Warfarin limits the synthesis of γ-glutamyl carboxylated forms of coagulation factors, factor II, factor VII, factor IX, and factor X, protein C, and protein S and as a result impairs the function of these proteins. In contrast, direct inhibitors of throm- bin only affect one enzyme in the coagulation cascade. The aim of this study was to investigate the antithrombotic effect and the slope of the dose- response curves of the multifactorial coagulation inhibitor warfarin in comparison with the single factor low-molecular-weight thrombin inhibitors melagatran and inogatran. An arterial thrombosis model in rats was used, and vessel damage was induced by topical application of ferric chloride to the carotid artery. The slopes of the dose-response curves were 3.6, 1.8, 1.1, and 1.2, for warfarin, heparin, inogatran, and melagatran, respectively. For warfarin the antithrombotic effect increased from 23% to 81% when the dose was doubled. In contrast, 10-fold increases in the doses of inogatran and melagatran were necessary to obtain a similar increase in antithrombotic effect. The doses needed to obtain 80% antithrombotic effect for heparin, warfarin, and melagatran were investigated in a tail transection bleeding model. For heparin, this dose significantly prolonged the bleeding time and the blood loss; for warfarin, only the total bleeding time was increased while for melagatran there was no increase in bleeding. We conclude that, thrombin inhibitors affecting only one enzyme in the coagulation cascade seem preferable to inhibitors affecting multiple enzymes, such as warfarin, due to shallower dose-response curves and a wider therapeutic interval.

Melagatran, an oral active-site inhibitor of thrombin, prevents or delays formation of electrically induced occlusive thrombus in the canine coronary artery.

Intravenous administration of thrombin inhibitors, such as hirudin, has been shown to decrease the frequency of coronary artery reocclusion after thrombolysis. However, recent findings in large clinical trials in patients with unstable angina and myocardial infarction have failed to demonstrate a sustained antithrombotic effect after cessation of drug treatment. These findings indicate a need for a prolonged antithrombotic regimen, preferably an orally active thrombin inhibitor. To test the hypothesis that a regimen consisting of oral thrombin inhibitor will delay or prevent the formation of occlusive clot, anesthetized dogs were given saline (n = 9) or a single dose of a novel active site low-molecular-weight thrombin inhibitor melagatran by nasogastric tube (1.5 mg/kg, n = 6; 2.5 mg/kg, n = 6), and 15 min later, a potent thrombogenic stimulus in the form of anodal current (100 microA) was applied to the intimal surface of the narrowed left anterior descending coronary artery (LAD). All saline-treated dogs developed stable thrombus, indicated by zero flow at 34 +/- 7 min after initiation of direct current. On the other hand, one of the six dogs given high-dose melagatran did not develop thrombotic occlusion of the LAD during the entire 4 h of observation. Mean time to occlusive thrombus formation in 11 other dogs was prolonged 4-5 times as compared with that in the saline-treated dogs (p < 0.001). Spontaneous thrombolysis was observed in three of 11 dogs after initial clot formation. Overall, the coronary artery was patent for 68% (low dose) and 75% (high dose) of the observation period in melagatran-treated dogs (vs. 14% of observation period in saline-treated dogs). Peak plasma concentration was 0.87 +/- 0.22 microM in dogs given low-dose and 1.38 +/- 0.30 microM in dogs given high-dose melagatran. The activated partial thromboplastin time (aPTT) increased 1.5-fold at peak plasma concentration of melagatran. These observations imply (a) thrombin generation plays a critical role in thrombus formation in narrowed coronary arteries, (b) oral melagatran prevents or delays thrombus formation, whereas the aPTT is only modestly prolonged, and (c) the thrombus formed in the presence of melagatran is prone to spontaneous lysis in this canine model of coronary thrombosis.

Antithrombotic Effect of Two Low Molecular Weight Thrombin Inhibitors and a Low-Molecular Weight Heparin in a Caval Vein Thrombosis Model in the Rat

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.