Elisabeth Perzborn

In vitro and in vivo studies of the novel antithrombotic agent BAY 59‐7939—an oral, direct Factor Xa inhibitor

Summary.  BAY 59‐7939 is an oral, direct Factor Xa (FXa) inhibitor in development for the prevention and treatment of arterial and venous thrombosis. BAY 59‐7939 competitively inhibits human FXa (Ki 0.4 nm) with > 10 000‐fold greater selectivity than for other serine proteases; it also inhibited prothrombinase activity (IC50 2.1 nm). BAY 59‐7939 inhibited endogenous FXa more potently in human and rabbit plasma (IC50 21 nm) than rat plasma (IC50 290 nm). It demonstrated anticoagulant effects in human plasma, doubling prothrombin time (PT) and activated partial thromboplastin time at 0.23 and 0.69 µm, respectively. In vivo, BAY 59‐7939 reduced venous thrombosis (fibrin‐rich, platelet‐poor thrombi) dose dependently (ED50 0.1 mg kg−1 i.v.) in a rat venous stasis model. BAY 59‐7939 reduced arterial (fibrin‐ and platelet‐rich) thrombus formation in an arteriovenous (AV) shunt in rats (ED50 5.0 mg kg−1 p.o.) and rabbits (ED50 0.6 mg kg−1 p.o.). Slight inhibition of FXa (32% at ED50) reduced thrombus formation in the venous model; to affect arterial thrombosis in the rat and rabbit, stronger inhibition of FXa (74%, 92% at ED50) was required. Calculated plasma levels in rabbits at the ED50 were 14‐fold lower than in the rat AV shunt model, correlating with the 14‐fold lower IC50 of FXa inhibition in rabbit compared with rat plasma; this may suggest a correlation between FXa inhibition and antithrombotic activity. Bleeding times in rats and rabbits were not significantly affected at antithrombotic doses (3 mg kg−1 p.o., AV shunt). Based on these results, BAY 59‐7939 was selected for clinical development.

The discovery and development of rivaroxaban, an oral, direct factor Xa inhibitor

The activated serine protease factor Xa is a promising target for new anticoagulants. After studies on naturally occurring factor Xa inhibitors indicated that such agents could be effective and safe, research focused on small-molecule direct inhibitors of factor Xa that might address the major clinical need for improved oral anticoagulants. In 2008, rivaroxaban (Xarelto; Bayer HealthCare) became the first such compound to be approved for clinical use. This article presents the history of rivaroxabans development, from the structure–activity relationship studies that led to its discovery to the preclinical and clinical studies, and also provides a brief overview of other oral anticoagulants in advanced clinical development.

In vitro inhibition of thrombin generation, after tissue factor pathway activation, by the oral, direct factor Xa inhibitor rivaroxaban

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Laboratory assessment of rivaroxaban: a review

Research into new anticoagulants for preventing and treating thromboembolic disorders has focused on targeting single enzymes in the coagulation cascade, particularly Factor Xa and thrombin, inhibition of which greatly decreases thrombin generation. Based on the results of phase III clinical trials, rivaroxaban, a direct Factor Xa inhibitor, has been approved in many countries for the management of several thromboembolic disorders. Owing to its predictable pharmacokinetic and pharmacodynamic characteristics, fixed-dose regimens are used without the need for routine coagulation monitoring. In situations where assessment of rivaroxaban exposure may be helpful, anti-Factor Xa chromogenic assays (in tandem with standard calibration curves generated with the use of rivaroxaban calibrators and controls) could be used. It is important to note that test results will be affected by the timing of blood sampling after rivaroxaban intake. In addition, the anti-Factor Xa method measures the drug concentration and not the intensity of the drug’s anticoagulant activity, and a higher than expected rivaroxaban plasma level does not necessarily indicate an increased risk of bleeding complications. Therefore, clinicians need to consider test results in relation to the pharmacokinetics of rivaroxaban and other patient risk factors associated with bleeding.

Evaluation of the Prothrombin Time for Measuring Rivaroxaban Plasma Concentrations Using Calibrators and Controls: Results of a Multicenter Field Trial

This study evaluated the prothrombin time (PT) assay for the measurement of plasma concentrations of rivaroxaban using calibrators and controls. The intra- and interlaboratory precision of the measurement was investigated in a field trial involving 21 laboratories. Each laboratory was provided with rivaroxaban calibrators and control plasma samples containing different concentrations of rivaroxaban, and PT reagents. The evaluation was carried out over 2 consecutive weeks using centrally provided and local PT reagents. A calibration curve was produced each day (for inter-run precision), and day-to-day precision was evaluated by testing 3 control plasma samples. A large interlaboratory variation (in seconds) was observed with local PT reagents. The results were less variable when expressed as rivaroxaban concentrations (ng/mL) or when central PT reagent was used (STA Neoplastine CI Plus). The widely available PT assay, in conjunction with rivaroxaban calibrators, may be useful for the measurement of peak plasma levels of rivaroxaban.

Measurement and Reversal of Prophylactic and Therapeutic Peak Levels of Rivaroxaban An In Vitro Study

Background: Rivaroxaban (Xarelto, Bayer HealthCare, Leverkusen, Germany) is a new oral anticoagulant drug. Anticoagulants may cause bleeding, thereby requiring reliable monitoring and efficient therapy. We investigated thromboelastometry versus routine coagulation tests to measure prophylactic and therapeutic concentrations of rivaroxaban and their reversal with prothrombin complex concentrate (PCC) and activated recombinant factor VII (rFVIIa) in vitro. Methods: Rivaroxaban was solubilized, and PCC and rFVIIa were added in 2 concentrations to the rivaroxaban-spiked blood samples, and thromboelastometry and measurements were performed. Results: Rivaroxaban increased tissue factor–activated clotting time (CTExTEM) dose dependently. Activated partial prothrombin time (aPTT), prothrombin time ratio (PTR), and prothrombin time (PT) were changed significantly in both concentrations. Reversal with PCC in both dosages caused no significant change in the measured parameters. For prophylactic rivaroxaban dosage, rFVIIa changed the PT significantly but not CTExTEM, aPTT, and PTR. For therapeutic rivaroxaban dosage, the CTExTEM was significantly reduced. The other parameters remained unaffected. Conclusions: Thromboelastometry can detect rivaroxaban effects. In vitro rFVIIa seems highly effective for reversal in contrast to PCC.

A role for coagulation factor Xa in experimental pulmonary arterial hypertension

AIMS Anticoagulation with warfarin is recommended for the treatment of patients with pulmonary arterial hypertension (PAH). However, the therapeutic benefit of anticoagulation has not yet been demonstrated experimentally or clinically. Here, rivaroxaban, an oral, direct factor Xa (FXa) inhibitor, was compared with warfarin and enoxaparin in the prevention of right ventricular (RV) dysfunction and hypertrophy in the monocrotaline (MCT) model of pulmonary hypertension. METHODS AND RESULTS Sprague-Dawley rats (n = 10 per group) were randomized to receive rivaroxaban, warfarin, enoxaparin, or placebo before receiving a subcutaneous injection of MCT 60 mg/kg or saline. Rivaroxaban and enoxaparin were administered for 28 days starting 4 h before MCT injection; warfarin was given for 35 days initiated 7 days before MCT injection. RV haemodynamics and hypertrophy were assessed 28 days after MCT administration. Rivaroxaban dose-dependently reduced systolic and end-diastolic RV pressure increase and RV hypertrophy. Warfarin reduced RV pressure increase only. Enoxaparin had no effect on either parameter. Severe bleeding occurred in four and five rats treated with warfarin and enoxaparin, respectively, whereas no overt bleeding was observed in rats treated with rivaroxaban. CONCLUSION Selective, direct inhibition of FXa by rivaroxaban effectively prevented RV dysfunction and hypertrophy in MCT-injected rats, indicating a role for coagulation factors in experimental pulmonary hypertension. Clinical investigation of the impact of early and continued administration of a specific FXa inhibitor such as rivaroxaban on the course of PAH should be considered.