FXa (IC50 = 0.7 nM); FXa (Ki = 0.4 nM)

An oral direct Factor Xa (FXa) inhibitor called rivaroxaban (BAY 59-7939) is being developed for the treatment and prevention of venous and arterial thrombosis. Rivaroxaban inhibits prothrombinase activity (IC50 2.1 nM) and human FXa (Ki 0.4 nM) competitively with selectivity that is >10,000 times better than that of other serine proteases. In comparison to rat plasma (IC50 290 nM), human and rabbit plasma exhibit a more effective inhibition of endogenous FXa by rivaroxaban (IC50 21 nM). In human plasma, it exhibits anticoagulant properties, activating partial thromboplastin time at 0.69 μM and increasing prothrombin time (PT)[2].

A strong and specific direct FXa inhibitor with good oral absorption and in vivo action is rivaroxaban (BAY 59-7939)[1]. When given as an intravenous bolus prior to thrombus induction, rivaroxaban (BAY 59-7939) decreases thrombus formation (ED50 0.1 mg/kg), suppresses FXa, and dose-dependently prolongs PT. At the ED50, there is a small change in PT and FXa (1.8-fold increase and 32% inhibition, respectively). At a dosage of 0.3 mg/kg, which virtually completely blocks thrombus formation, rivaroxaban exhibits a moderate prolongation of PT (3.2±0.5-fold) and a suppression of FXa activity (65±3%)[2].

In Vitro Studies:[1]
FXa and Related Serine Proteases. The enzymatic activity against human FVIIa, FIXa, FXa, FXIa, thrombin, plasmin, trypsin, urokinase, and activated protein C was measured using chromogenic or fluorogenic substrates in 96-well microtiter plates at 25 °C. The enzymes were incubated with the test compound or its solvent (DMSO) for 10 min, and the reactions were initiated by the addition of the appropriate substrate. Color change was monitored continuously at 405 nm by a Spectra Rainbow Thermo Reader, and fluorescence was measured at 360/465 nm by a SPECTRAFluor Plus microplate reader. Substrates and enzymes were dissolved in aqua bidest or the appropriate assay buffer.

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Prothrombin Time (PT) Assay.[1]
Commercially available kits were used to measure PT. Clotting times were measured in a coagulometer, according to the manufacturer's instructions. Increasing concentrations of inhibitor or solvent were added to plasma and incubated for 10 min at 37 °C. Clotting times were measured and compared with those from the appropriate control plasma.

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In VitroPlasma-Protein Binding. [1]
In vitro plasma-protein binding was evaluated by an equilibrium dialysis method (Scholtan 1962). [14C]-5 was added to each aliquot of rat, dog, and human plasma to make target concentrations of 0.1, 1.0, 3.0, 10, 30, and 100 mg L-1; in addition, a target concentration of 400 mg L-1 was prepared, but only for human plasma. After incubation for 15 min at 37 °C, 0.8 mL of the spiked plasma was dialyzed with an equal volume of phosphate-buffered isotonic solution (PBS, pH 7.4) for 1 h at 37 °C in an equilibrium dialyzer equipped with 0.8 mL Teflon half cells separated by a cellulose membrane (Diachema 10.14 cellulose membrane, MWCO 5000 kDa; Dianorm GmbH). The radioactivity of [14C]-5 in the buffer and the plasma was determined by LSC. The fraction of unbound 5 (fu [%]) was calculated as follows:  fu = cu/c × 100, where cu is the concentration of unbound 5 and c is the total concentration of 5.

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X-ray Crystallography.[1]
The X-ray crystal structure of 5 in complex with human FXa was performed by Proteros Biostructures GmbH in Planegg-Martinsried, Germany. Crystals of human FXa in complex with 5 were prepared as described with small modifications. Synchrotron data to 2.08 Å were collected at the Swiss Light Source (SLS) in Villigen at 100 K. Data were integrated, scaled, and merged using XDS. Structure solution was done by molecular replacement using 1EQZ coordinates from PDB as the search model. The model was refined with CNX, using data between 20.0 and 2.08 Å. Diffraction data statistics and structure parameters are shown in Table 7.

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Enzyme assays[2]
The activity of BAY 59-7939 against purified serine proteases was measured using chromogenic or fluorogenic substrates in 96-well microtiter plates at 25 °C. The enzymes were incubated with BAY 59-7939 or its solvent, dimethyl sulfoxide (DMSO), for 10 min. The reactions were initiated by the addition of the substrate, and the color or fluorescence was monitored continuously at 405 nm using a Spectra Rainbow Thermo Reader, or at 630/465 nm using a SPECTRAfluor plus, respectively, for 20 min (if not otherwise stated).

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Enzymatic activity was 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 50 mm 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 h as described previously. 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, was started by the addition of I-1100 (50 µm), and measured for 60 min.

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FXa activity in plasma [2]
Human, rat, or rabbit plasma (45 µL) was mixed with 5 µL hirudin (10 µg mL−1), 5 µL BAY 59-7939 or DMSO, and 50 µL RVV (human, 0.7 mU mL−1; rat/rabbit, 3.5 mU mL−1), dissolved in 50 µm CaCl2 at 37 °C. Chromozym X (50 µL; 600 µm) was added after 15 min. The increase in optical density was measured at 37 °C, as described above.

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Coagulation assays[2]
Activated partial thromboplastin time (aPTT) and prothrombin time (PT) were measured using commercially available kits. BAY 59-7939 or DMSO (3 µL) were added to 100 µL platelet-poor plasma (PPP) and incubated for 10 min at 37 °C. Clotting times were measured in a coagulometer (Biomatic 4000), in accordance with the manufacturer's instructions (final volume 303 µL). Anticoagulant activity was defined as the concentration required to double the plasma clotting times [CT2 (µm)].

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Plasma preparation[2]
Human blood was collected by venipuncture from healthy subjects who had not been medicated during the last 10 days. Rabbit blood was obtained by puncture of the A. carotis, and rat blood was withdrawn from the abdominal aorta under anesthesia. Blood was collected into plastic tubes containing 1/10 volume of 3.8% trisodium citrate. PPP was obtained by immediate centrifugation at 2500 g for 10 min at 4 °C, and stored at − 20 °C.

Dssolved in polyethylene glycol/H2O/ glycerol (996 g/100 g/60 g) (for i.v.); and dissolved in solutol/ethanol/H2O [40%/10%/50% (v/v/v)] (for p.o.);
≤0.3 mg/kg for both i.v. and for p.o.; i.v. injection or Oral gavage;
Fasted, male Wistar rats (HsdCpb:WU) and fasted, female New Zealand White rabbits (Esd:NZW).

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In Vivo Studies: [1]
Arteriovenous (AV) Shunt Model. The antithrombotic activity was determined in an AV shunt in anesthetized rats, as described previously 21 with minor modifications:  The right common carotid artery and the left jugular vein were cannulated with two 100 mm-long, saline-filled, polyethylene catheters. The catheters were connected with a 30 mm-long polyethylene tube containing a rough nylon thread (40 mm × 0.15 mm), folded to create a 20 mm-long double string. The test compound dissolved in poly(ethylene glycol)/water/glycerol (996 g/100 g/60 g) or vehicle was given by intravenous bolus injection into a tail vein 10 min before thrombus induction. Alternatively, the test compound dissolved in solutol/ethanol/water (40%/10%/50% [v/v/v]) or vehicle was administered orally 90 min before thrombus induction. The shunt was opened for 15 min, and the nylon thread covered with the thrombus was then withdrawn and weighed. Blood samples were withdrawn from the carotid artery just after thrombus removal.

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Rat venous stasis model [2]
Thrombus formation was induced in anesthetized rats (n = 10 per dose group) as described previously, with minor modifications. The abdominal vena cava was exposed and two loose sutures (8–10 mm apart) were placed below the left renal venous branch. BAY 59-7939 dissolved in polyethylene glycol/H2O/glycerol (996 g/100 g/60 g), or vehicle was given by intravenous (i.v.) bolus injection into a tail vein 15 min before thrombus induction. Thromboplastin (0.5 mg kg−1) was injected into a femoral vein and, after 15 s, the proximal and distal sutures were tied. Fifteen minutes later, the ligated segment was removed, the thrombus withdrawn and weighed. Blood samples were obtained by cardiac puncture immediately before thrombus removal.

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Arteriovenous shunt model in rats and rabbits [2]
An arteriovenous (AV) shunt in anesthetized rats and rabbits was performed as described previously, with minor modifications. The right common carotid artery and the left jugular vein were cannulated with two 100-mm-long, saline-filled catheters. In rats (n = 10 per dose group), the polyethylene catheters were connected with a 30-mm-long polyethylene tube containing a rough nylon thread (40 × 0.15 mm), folded into a double string. In rabbits (n = 6 per dose group), polyurethane vein catheters (outside diameter 2.1 mm) were connected with a 40-mm-long polyethylene tube, containing a rough nylon thread (60 × 0.15 mm), folded into a double string. BAY 59-7939, dissolved in solutol/ethanol/H2O [40%/10%/50% (v/v/v)], or vehicle was given orally 90 min before the shunt was opened for 15 min. The nylon thread was then withdrawn and weighed. Blood samples were withdrawn from the carotid artery just after thrombus removal.

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Rat tail-bleeding model[2]
BAY 59-7939 (n = 10 per dose group) or vehicle was given orally 90 min before the tails of anesthetized rats were transected 2 mm from the tip and vertically immersed in saline at 37 °C. The time until continuous blood flow ceased for > 30 s was measured, with a maximum observation time of 10 min (longer bleeding times were assigned a value of 10 min).

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Rabbit ear-bleeding model[2]
Ear-bleeding time (EBT) was determined in anesthetized rabbits (n = 5 per dose group), as described previously. A standardized 3-mm-long incision was made at different sites of the right ear in each animal 90 and 105 min after administration of oral BAY 59-7939 or vehicle. Blood from the incision was removed with filter paper every 30 s. The time until the bleeding stopped was measured.

Absorption, Distribution and Excretion
Following oral administration, rivaroxaban is rapidly absorbed and reaches peak plasma concentration in 2-4 hours. Bioavailability of the 10 mg dose is >80%. However, the 15-20 mg dose have a lower bioavailability if taken in the fasted state and consequently should be taken with food.
Approximately two-thirds of rivaroxaban is excreted into urine (via active tubular secretion in which approximately 36% as unchanged drug and 30% as inactive metabolism). The remaining third of the administered dose is excreted via feces in which 7% is in the form of unchanged drug and 21% as inactive metabolites.
The steady state Vd is 50 L
Systemic clearance is approximately 10 L/h, so rivaroxaban is considered a drug with low clearance. Renal clearance is ~3-4 L/h.
Following oral administration, approximately one-third of the absorbed dose is excreted unchanged in the urine, with the remaining two-thirds excreted as inactive metabolites in both the urine and feces. In a Phase 1 study, following the administration of a (14)C-rivaroxaban dose, 66% of the radioactive dose was recovered in urine (36% as unchanged drug) and 28% was recovered in feces (7% as unchanged drug). Unchanged drug is excreted into urine, mainly via active tubular secretion and to a lesser extent via glomerular filtration (approximate 5:1 ratio). Rivaroxaban is a substrate of the efflux transporter proteins P-gp and ABCG2 (also abbreviated Bcrp). Rivaroxaban's affinity for influx transporter proteins is unknown.
Plasma protein binding of rivaroxaban in human plasma is approximately 92% to 95%, with albumin being the main binding component. The steady-state volume of distribution in healthy subjects is approximately 50 L.
Absorption of rivaroxaban is dependent on the site of drug release in the GI tract. A 29% and 56% decrease in AUC and Cmax compared to tablet was reported when rivaroxaban granulate is released in the proximal small intestine. Exposure is further reduced when drug is released in the distal small intestine, or ascending colon. Avoid administration of rivaroxaban distal to the stomach which can result in reduced absorption and related drug exposure.
The absolute bioavailability of rivaroxaban is dose-dependent. For the 10 mg dose, it is estimated to be 80% to 100% and is not affected by food. Xarelto 10 mg tablets can be taken with or without food. For the 20 mg dose in the fasted state, the absolute bioavailability is approximately 66%. Coadministration of Xarelto with food increases the bioavailability of the 20 mg dose (mean AUC and Cmax increasing by 39% and 76% respectively with food). Xarelto 15 mg and 20 mg tablets should be taken with food.
For more Absorption, Distribution and Excretion (Complete) data for Rivaroxaban (8 total), please visit the HSDB record page.

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Metabolism / Metabolites
Approximately two-thirds of the dose is metabolized. It is metabolized by CYP3A4, CYP3A5, CYP2J2 and CYP-independant mechanisms
Rivaroxaban undergoes oxidative degradation by cytochrome P-450 (CYP) isoenzymes 3A4/5 and 2J2 and hydrolysis; metabolites are subsequently eliminated through renal and fecal/biliary routes. No major circulating metabolites have been identified in plasma.

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Biological Half-Life
The terminal half life is 5-9 hours in adults and 11-13 hours in the elderly.
The terminal elimination half-life is 11 to 13 hours in the elderly.
The terminal elimination half-life of rivaroxaban is 5 to 9 hours in healthy subjects aged 20 to 45 years.

Hepatotoxicity
Chronic therapy with rivaroxaban is associated with moderate ALT elevations (greater than 3 times the upper limit of normal) in 1.5% to 3% of patients, an overall rate which is slightly lower than with low molecular weight heparins and similar to the rates with warfarin. During the large, prelicensure clinical trials of rivaroxaban, several instances of ALT elevations with jaundice occurred, but few details were provided and it was not clear whether the liver injury was clinically apparent. The cases were evidently mild and self-limited, resolving completely once therapy was stopped. Since its licensure and more wide scale use, rivaroxaban has been linked to many instances of acute liver injury with jaundice. The clinical features of these cases varied widely. Most cases had an onset within 1 to 8 weeks of starting rivaroxaban and presented with jaundice, fatigue and a hepatocellular pattern of serum enzyme elevations. In some individuals, a cholestatic or mixed pattern was found. Immunoallergic features and autoimmune markers were atypical but at least one case occurred with skin rash and fever suggestive of DRESS syndrome. One case of acute hepatic necrosis and death attributed to rivaroxaban has been reported, but ischemic hepatitis due to severe heart failure was a more likely cause of the acute liver failure. All other reported cases of rivaroxaban induced liver injury recovered upon stopping rivaroxaban, usually quite promptly, within 2 to 4 weeks. In large health care databases, hospitalization for acute liver injury arises in approximately 1 in 2,200 cases, but whether all cases in these databases represent liver injury from rivaroxaban is uncertain.
Likelihood score: A (well established cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Several case reports and one thorough pharmacokinetic analysis consistently indicate that maternal doses of rivaroxaban of 15 to 30 mg daily produce low levels in milk that are considerably below doses (<2%) required for anticoagulation in infants. Plasma rivaroxaban levels in two breastfed infants were undetectable. If the mother requires rivaroxaban, it is not a reason to discontinue breastfeeding.
◉ Effects in Breastfed Infants
A 38-year-old woman with antiphospholipid syndrome began rivaroxaban 15 mg (0.19 mg/kg) daily at 5 days postpartum for prophylaxis of deep vein thrombosis. She partially breast-fed her infant (at least 50%). No apparent evidence of bleeding was noted in the infant at 1- and 3-month check-ups and development was normal at 18 months of age.
Two mothers received rivaroxaban 15 mg (0.22 and 0.25 mg/kg) daily beginning 3 days postpartum for prophylaxis of deep vein thrombosis. At 3 months postpartum, their infants continued to be breastfed (extent not stated) and had no health problems or bleeding events.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Plasma protein binding is about 92% to 95%

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Interactions
Patients with renal impairment receiving full dose Xarelto in combination with drugs classified as combined P-gp and weak or moderate CYP3A4 inhibitors (e.g., amiodarone, diltiazem, verapamil, quinidine, ranolazine, dronedarone, felodipine, erythromycin, and azithromycin) may have increases in exposure compared with patients with normal renal function and no inhibitor use, since both pathways of rivaroxaban elimination are affected. Xarelto should be used in patients with CrCl 15 to 50 mL/min who are receiving concomitant combined P-gp and weak or moderate CYP3A4 inhibitors only if the potential benefit justifies the potential risk.
Single doses of enoxaparin and Xarelto given concomitantly resulted in an additive effect on anti-factor Xa activity. Single doses of warfarin and Xarelto resulted in an additive effect on factor Xa inhibition and PT. Concomitant aspirin use has been identified as an independent risk factor for major bleeding in efficacy trials. NSAIDs are known to increase bleeding, and bleeding risk may be increased when NSAIDs are used concomitantly with Xarelto. Coadministration of the platelet aggregation inhibitor clopidogrel and Xarelto resulted in an increase in bleeding time for some subjects. Avoid concurrent use of Xarelto with other anticoagulants due to increased bleeding risk unless benefit outweighs risk. Promptly evaluate any signs or symptoms of blood loss if patients are treated concomitantly with aspirin, other platelet aggregation inhibitors, or NSAIDs.
Results from drug interaction studies and population PK analyses from clinical studies indicate coadministration of Xarelto with a combined P-gp and strong CYP3A4 inducer (e.g., rifampicin, phenytoin) decreased rivaroxaban exposure by up to 50%. Similar decreases in pharmacodynamic effects were also observed. These decreases in exposure to rivaroxaban may decrease efficacy. Avoid concomitant use of Xarelto with drugs that are combined P-gp and strong CYP3A4 inducers (e.g., carbamazepine, phenytoin, rifampin, St. John's wort).
When data suggest a change in exposure is unlikely to affect bleeding risk (e.g., clarithromycin, erythromycin), no precautions are necessary during coadministration with drugs that are combined P-gp and CYP3A4 inhibitors. Avoid concomitant administration of Xarelto with combined P-gp and strong CYP3A4 inhibitors.
For more Interactions (Complete) data for Rivaroxaban (10 total), please visit the HSDB record page.

[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)
[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.

Therapeutic Uses
Anticoagulant
Xarelto is indicated for the treatment of deep vein thrombosis (DVT). /Included in US product label/
Xarelto is indicated to reduce the risk of stroke and systemic embolism in patients with nonvalvular atrial fibrillation. There are limited data on the relative effectiveness of Xarelto and warfarin in reducing the risk of stroke and systemic embolism when warfarin therapy is well-controlled. /Included in US product label/
Xarelto is indicated for the prophylaxis of deep vein thrombosis, which may lead to pulmonary embolism in patients undergoing knee or hip replacement surgery. /Included in US product label/
For more Therapeutic Uses (Complete) data for Rivaroxaban (7 total), please visit the HSDB record page.

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Drug Warnings
/BOXED WARNING/ WARNING: PREMATURE DISCONTINUATION OF XARELTO INCREASES THE RISK OF THROMBOTIC EVENTS. Premature discontinuation of any oral anticoagulant, including Xarelto, increases the risk of thrombotic events. If anticoagulation with Xarelto is discontinued for a reason other than pathological bleeding or completion of a course of therapy, consider coverage with another anticoagulant
/BOXED WARNING/ WARNING: SPINAL/EPIDURAL HEMATOMA. Epidural or spinal hematomas have occurred in patients treated with Xarelto who are receiving neuraxial anesthesia or undergoing spinal puncture. These hematomas may result in long-term or permanent paralysis. Consider these risks when scheduling patients for spinal procedures. Factors that can increase the risk of developing epidural or spinal hematomas in these patients include: use of indwelling epidural catheters; concomitant use of other drugs that affect hemostasis, such as non-steroidal anti-inflammatory drugs (NSAIDs), platelet inhibitors, other anticoagulants; a history of traumatic or repeated epidural or spinal punctures a history of spinal deformity or spinal surgery. Monitor patients frequently for signs and symptoms of neurological impairment. If neurological compromise is noted, urgent treatment is necessary. Consider the benefits and risks before neuraxial intervention in patients anticoagulated or to be anticoagulated for thromboprophylaxis
Rivaroxaban increases the risk of hemorrhage and can cause serious or fatal bleeding. Bleeding complications were the most common adverse effects of rivaroxaban reported in clinical trials.
Use of rivaroxaban should be avoided in patients with moderate (Child-Pugh class B) or severe (Child-Pugh class C) hepatic impairment or with any hepatic disease associated with coagulopathy; systemic exposure and risk of bleeding may be increased in such patients.
For more Drug Warnings (Complete) data for Rivaroxaban (13 total), please visit the HSDB record page.

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Pharmacodynamics
Rivaroxaban is an anticoagulant which binds directly to factor Xa. Thereafter, it effectively blocks the amplification of the coagulation cascade, preventing the formation of thrombus. Rivaroxaban is a unqiue anticoagulant for two reasons. First of all, it is does not involve antithrombin III (ATIII) to exert its anticoagulant effects. Secondly, it is an oral agent whereas the widely used unfractionated heparin and low molecular weight heparins are for parenteral use only. Although the activated partial thromboplastin time (aPTT) and HepTest (a test developed to assay low molecular weight heparins) are prolonged in a dose-dependant manner, neither test is recommended for the assessment of the pharmacodynamic effects of rivaroxaban. Anti-Xa activity and inhibition of anti-Xa activity monitoring is also not recommended despite being influenced by rivaroxaban.

C19H18CLN3O5S

435.88

435.065

C, 52.35; H, 4.16; Cl, 8.13; N, 9.64; O, 18.35; S, 7.36

366789-02-8

Rivaroxaban-d4;1132681-38-9

9875401

White to off-white solid powder

1.5±0.1 g/cm3

732.6±60.0 °C at 760 mmHg

228-229°C

396.9±32.9 °C

0.0±2.4 mmHg at 25°C

1.633

1.84

1

6

5

29

645

1

C1COCC(=O)N1C2=CC=C(C=C2)N3C[C@@H](OC3=O)CNC(=O)C4=CC=C(S4)Cl

KGFYHTZWPPHNLQ-AWEZNQCLSA-N

InChI=1S/C19H18ClN3O5S/c20-16-6-5-15(29-16)18(25)21-9-14-10-23(19(26)28-14)13-3-1-12(2-4-13)22-7-8-27-11-17(22)24/h1-6,14H,7-11H2,(H,21,25)/t14-/m0/s1

(S)-5-chloro-N-((2-oxo-3-(4-(3-oxomorpholino)phenyl)oxazolidin-5-yl)methyl)thiophene-2-carboxamide

BAY 59-7939; Rivaroxaban; BAY59-7939; BAY-59-7939; trade name: Xarelto.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 2.5 mg/mL (5.74 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.74 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 0.5% methylcellulose+0.2% Tween 80:5 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)