Rivaroxaban (Xarelto, BAY 59-7939) is a direct inhibitor of Factor Xa with Ki and IC50 of 0.4 nM and 0.7 nM, respectively.
Targets
Factor Xa
IC50
0.7 nM
In Vitro
Rivaroxaban is an oral, direct inhibitor of Factor Xa (FXa), being developed for the prevention and treatment of arterial and venous thrombosis with a Ki of 0.4 nM. Rivaroxaban also inhibits prothrombinase activity with IC50 of 2.1 nM. Rivaroxaban also shows a similar affinity to purified human and rabbit FXa (IC50 0.7 nM and 0.8 nM, respectively), but a lesser potency against purified rat FXa (IC50 3.4 nM). Endogenous human and rabbit FXa in plasma is inhibited to a similar extent by Rivaroxaban (IC50 21 nM and 21 nM, respectively), while 14-fold higher concentrations are required in rat plasma (IC50 290 nM). [1] Rivaroxaban exhibits high permeability and polarized transport across Caco-2 cells as a substrate of the P-gp, but exhibits no inhibitory effect on P-gp-mediated drug transport up to concentrations of 100 μM in vitro. [2]
In Vivo
Rivaroxaban reduces venous thrombosis in a dose dependent manner (ED50 0.1 mg/kg i.v.) in a rat venous stasis model. Rivaroxaban reduces arterial thrombus formation in an arteriovenous (AV) shunt in rats (ED50 5.0 mg/kg p.o.) and rabbits (ED50 0.6 mg/kg p.o.). [1] Plasma pharmacokinetics of Rivaroxaban are linear across the investigated dose range (1-10 mg/kg in rats, 0.3-3 mg/kg in dogs). Plasma clearance is low: 0.4 L/kg/h in rats and 0.3 L/kg/h in dogs; the volume of distribution (V(ss)) is moderate: 0.3 L/kg in rats, and 0.4 L/kg in dogs. The elimination half-life after oral administration is short in both species (0.9-2.3 hours). [3]
Clinical Trials
Rivaroxaban is currently being tested in Phase II clinical trials in patients with cardiovascular disease.
The activity of Rivaroxaban against purified serine proteases is measured using chromogenic or fluorogenic substrates in 96-well microtiter plates. The enzymes are incubated with Rivaroxaban or its solvent, dimethyl sulfoxide (DMSO), for 10 minutes. 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 minutes. 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 chromozym trypsin (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-benzylsulfonamide-H2O (100 μM) and measured for 3 hours. The FIXaβ/FX assay, comprising FIXaβ (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 minutes. The inhibitory constant (Ki) against FXa is calculated according to the Cheng–Prusoff equation. The IC50 is the amount of inhibitor required to diminish the initial velocity of the control by 50%.
Caco-2, wild-type, and P-gp-overexpressing LLC-PK1
Concentrations
0 - 100 μM
Incubation Time
2 hours
Methods
LLC-PK1 and L-MDR1 cells are seeded in 96-well culture plates with microporous polycarbonate inserts and grown for 4 days in the same medium as used for cell cultures but without vincristine. The medium is replaced every 2 days. Before running the assay, the culture medium is replaced by HBSS buffer supplemented with 10 mM HEPES. Rivaroxaban are dissolved in DMSO and diluted with transport buffer to the respective final test concentrations (final DMSO concentration is always 1%). For inhibitor studies, the inhibitor is added at the appropriate concentration. counted. After 2 hour incubation at 37 °C, samples are taken from both compartments and, after the addition of ammonium acetate buffer and acetonitrile, are analyzed by LC-MS/MS.
Fasted, male Wistar rats (HsdCpb:WU) and fasted, female New Zealand White rabbits (Esd:NZW).
Formulation
Rivaroxaban dissolves in polyethylene glycol/H2O/ glycerol (996 g/100 g/60 g) and is given by i.v.Rivaroxaban dissolved in solutol/ethanol/H2O [40%/10%/50% (v/v/v)] and is given by p.o.
