Margareta Elg

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

UNLABELLED 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 alpha-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.

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.

Effect of activated prothrombin complex concentrate or recombinant factor VIIa on the bleeding time and thrombus formation during anticoagulation with a direct thrombin inhibitor.

UNLABELLED Melagatran is the active form of the oral, direct thrombin inhibitor H 376/95. In several animal models of thrombosis, the antithrombotic properties of melagatran have been demonstrated, without any increase in experimental bleeding. However, as with all anticoagulants, in emergency situations, reversal of the anticoagulation may be necessary. In this study, increasing doses of activated prothrombin complex concentrate (APCC, Feiba) or recombinant factor VIIa (r-F VIIa, NovoSeven) were superimposed on high doses of melagatran, or saline, in anaesthetised rats. The haemostatic effect was evaluated in two bleeding time models and a potential prothrombotic effect was evaluated in an arterial thrombosis model. Compared with melagatran alone (0.5 micromol/kg/h), Feiba in doses of > or =25 U/kg significantly shortened the prolonged bleeding time and reduced blood loss. In addition, Feiba > or =50 U/kg when added to melagatran (2 micromol/kg/h), significantly reduced bleeding time. No potentiation of thrombus formation was observed when Feiba was added to melagatran, compared with controls. NovoSeven at high doses (2-10 mg/kg) produced a nonsignificant trend in reduction of blood loss and with the highest dose (10 mg/kg) producing only a mild nonsignificant reduction in bleeding time. The prolonged prothrombin time (PT) and the ecarin clotting time (ECT) were more effectively shortened by Feiba than by NovoSeven. In contrast, whole blood clotting time (WBCT) was more effectively shortened by NovoSeven than by Feiba. Activated partial thromboplastin time (APTT) was shortened by NovoSeven but was prolonged by Feiba. Thrombin-antithrombin (TAT) complex formation was increased in a dose-dependent fashion more effectively by Feiba than by NovoSeven. CONCLUSION Feiba (APCC) reversed prolonged bleeding time and blood loss in rats treated with high doses of melagatran and compared with the control group thrombus formation was not potentiated. NovoSeven (r-F VIIa) at high doses had less pronounced effects on blood loss and bleeding times compared with Feiba.

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.

Effects of Agents, Used to Treat Bleeding Disorders, on Bleeding Time Prolonged by a Very High Dose of a Direct Thrombin Inhibitor in Anesthesized Rats and Rabbits

Melagatran is the active form of the oral, direct thrombin inhibitor, H 376/95, that is under evaluation in clinical trials for the prevention and treatment of thromboembolism. In this study, a single dose, calculated on body weight basis, of antifibrinolytic treatment, factor VIIa, factor VIII with and without von Willebrand factor (vWF), factor IX, activated (APCC) or nonactivated (PCC) prothrombin complex concentrates was given intravenously to rats and rabbits, in an attempt to reverse the prolonged bleeding time during intensive anticoagulation with melagatran (2 micromol/kg/h). The doses used were at or above human therapeutic doses. The cutaneous tail bleeding time in the rat, as well as the ear incision bleeding time and cuticle bleeding time, and the blood loss in the rabbit were used for evaluation of the hemostatic effects of these agents. In vivo Feiba (APCC) and Prothromplex-T (PCC) shortened the prolonged cutaneous bleeding times in rats (P<.05); Feiba and Autoplex (APCC) shortened the cutaneous bleeding times in rabbits (P<.05). In contrast, Prothromplex-T prolonged bleeding times and blood loss in the rabbits (P<.05). Ex vivo Feiba, Autoplex and NovoSeven (rF VIIa) significantly (P<.05) shortened the prolonged whole blood clotting time (WBCT). Prothromplex-T significantly prolonged WBCT, activated clotting time (ACT) and activated partial thromboplastin time (APTT). Feiba, Autoplex, and Prothromplex-T increased thrombin generation measured as increased thrombin-antithrombin complex (TAT) formation. In conclusion, APCCs were found to be the most effective agents for reversing bleeding time induced by a very high plasma concentration of melagatran. APCC and recombinant activated factor FVII (rF VIIa) effectively shortened the prolonged WBCT. Thus, stimulating thrombin generation with the use of APCC may counteract the anticoagulant effect observed with a very high dose of a thrombin inhibitor.

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.

Effects of inogatran, a new low-molecular-weight thrombin inhibitor, in rat models of venous and arterial thrombosis, thrombolysis and bleeding time

Inogatran (MW 439 Da), a new, selective, active site inhibitor of thrombin, was evaluated in three rat models of thrombosis. In the venous thrombosis model, inogatran dose-dependently inhibited thrombus formation with a > 80% antithrombotic effect at a plasma concentration of 0.45 μmol 1–1. In the arterial thrombosis model, inogatran dose-dependently inhibited thrombus formation, preserved vessel patency and the mean blood flow. Acetylsalicylic acid (ASA) potentiated the effects of low plasma concentrations of inogatran in the arterial thrombosis model. In the model of rt-PA-induced thrombolysis of a thrombus in the carotid artery, inogatran improved the patency time and the cumulative blood flow during the two hour thrombolysis period more than rt-PA alone. At high therapeutic plasma concentration of inogatran, there was only a moderate prolongation of bleeding time compared with the control value. It is concluded that inogatran is an effective antithrombotic agent both in the venous and arterial thrombosis models and also as adjuvant to rt-PA in the thrombolysis model.

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.

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

ABSTRACT

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).