The activity of Rivaroxaban (BAY 59-7939) against purified serine proteases is measured using chromogenic or fluorogenic substrates in 96-well microtiter plates at 25°C. The enzymes are incubated with Rivaroxaban or its solvent, DMSO, for 10 min. The reactions are initiated by the addition of the substrate, and the color or fluorescence is monitored continuously at 405 nm using a Spectra Rainbow Thermo Reader, or at 630/465 nm using a SPECTRAfluor plus, respectively, for 20 min. Enzymatic activity is analyzed in the following buffers (final concentrations): human FXa (0.5 nM), rabbit FXa (2 nM), rat FXa (10 nM), or urokinase (4 nM) in 50 mM Tris-HCl buffer, pH 8.3, 150 mM NaCl, and 0.1% bovine serum albumin (BSA); Pefachrome FXa (50-800 μM) or chromozym U (250 μM) with thrombin (0.69 nM), trypsin (2.2 nM), or plasmin (3.2 nM) in 0.1 μM Tris-HCl, pH 8.0, and 20 mM CaCl₂; chromozym TH (200 μM), chromozym plasmin (500 μM), or chromozymtrypsin (500 μM) with FXIa (1 nM) or APC (10 nM) in 50mM phosphate buffer, pH 7.4, 150 mM NaCl; and S 2366 (150 or 500 μM) with FVIIa (1 nM) and tissue factor (3 nM) in 50 mM Tris-HCl buffer,pH 8.0, 100 mM NaCl, 5 mM CaCl₂ and 0.3% BSA, H-D-Phe-Pro-Arg-6-amino-1-naphthalene-benzylsulfon-amide H₂O (100 μM) and measured for 3 h. The FIXab/FX assay, comprising FIXab (8.8 nM) and FX (9.5 nM) in 50 mM Tris-HCl buffer, pH 7.4, 100 mM NaCl, 5 mM CaCl₂ and 0.1% BSA, is started by the addition of I-1100 (50 μM), and measured for 60 min. The inhibitory constant (K_i) against FXa is calculated according to the Cheng-Prusoff equation (K_i=IC₅₀/1+[S]/K_m), where [S] is the substrate concentration, and K_m is the Michaelis-Menten constant. K_m is determined from a Lineweaver-Burk plot. The IC₅₀ is the amount of inhibitor required to diminish the initial velocity of the control by 50%^[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Rats^[2]
Fasted, male Wistar rats (HsdCpb:WU) are used. Rat venous stasis model Thrombus formation is induced in anesthetized rats (n=10 per dose group), with minor modifications. The abdominal vena cava is exposed and two loose sutures (8-10 mm apart) are placed below the left renal venous branch. Rivaroxaban dissolved in polyethylene glycol/H₂O/glycerol (996 g/100 g/60 g), or vehicle is given by intravenous (i.v.) bolus injection into a tail vein 15 min before thrombus induction. Thromboplastin (0.5 mg/kg) is injected into a femoral vein and, after 15 s, the proximal and distal sutures are tied. Fifteen minutes later, the ligated segment is removed, the thrombus withdrawn and weighed. Blood samples are obtained by cardiac puncture immediately before thrombus removal.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Roehrig S, et al. Discovery of the novel antithrombotic agent 5-chloro-N-({(5S)-2-oxo-3- [4-(3-oxomorpholin-4-yl)phenyl]-1,3-oxazolidin-5-yl}methyl)thiophene- 2-carboxamide (BAY 59-7939): an oral, direct factor Xa inhibitor. J Med Chem. 2005 Sep 22;48(19)  [Content Brief]

  [2]. Perzborn E, et al. In vitro and in vivo studies of the novel antithrombotic agent BAY 59-7939--an oral, direct Factor Xa inhibitor. J Thromb Haemost. 2005 Mar;3(3):514-21.  [Content Brief]