Impurity of Edoxaban

Human plasma's PT, TT, and APTT are prolonged by edoxaban in a concentration-dependent manner (1, 1, and 5 minutes, respectively)[1]. With an IC50 of 2.90 µM, edoxaban prevents platelet aggregation caused by thrombin[1].

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Inhibitory effect of Edoxaban/DU‐176b on FXa Inhibition of human FXa by DU‐176b was concentration‐dependent and competitive, as shown by the Lineweaver–Burk plot (Fig. 2). The Ki value was 0.561 nm (Table 1), a marked improvement in potency compared with DX‐9065a (Ki = 41 nm) [6]. DU‐176b also inhibited cynomolgus monkey and rabbit FXa with similar potency, whereas the Ki for rat FXa was higher than that for human FXa (Table 1), similar to the profile of DX‐9065a. For FXa bound to FVa, Ca2+ and phospholipids within the prothrombinase complex using S‐2222 as a substrate, inhibition by DU‐176b was competitive (Fig. 3A). The Ki value was 0.903 nm, comparable to its inhibition of free FXa. DU‐176b also suppressed the generation of thrombin from prothrombin by prothrombinase in a non‐competitive/mixed‐type of inhibition (Fig. 3B), with a 5.3‐fold higher Ki (2.98 nm) than that obtained with free FXa.

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Specificity of Edoxaban/DU‐176b DU‐176b was a weak inhibitor of thrombin and FIXa, with Ki values of 6.00 and 41.7 μm, respectively; more than 10 000‐fold higher than the Ki for FXa. There was no effect on the activities of FVIIa/sTF, FXIa, tPA, aPC, trypsin, plasmin and chymotrypsin, demonstrating the high specificity of DU‐176b for FXa.

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Anticoagulant activity in vitro PT, APTT and TT of human plasma were prolonged by Edoxaban/DU‐176b in a concentration‐dependent manner, doubling PT and APTT at 0.256 and 0.508 μm, respectively (Table 2). The CT2 for TT, however, was much higher (4.95 μm), reflecting its anti‐thrombin activity as shown in the enzyme inhibition assay. The potency of DU‐176b for PT prolongation was similar in human, cynomolgus monkey and rabbit plasma, whereas a higher concentration was needed in rat plasma.

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Effects on human platelet aggregation in vitro Edoxaban/DU‐176b did not impair human platelet aggregation induced by ADP, collagen or U46619 (a thromboxane A2 receptor agonist) at concentrations of up to 100 μm in PRP. Thrombin‐induced platelet aggregation was inhibited by a high concentration of DU‐176b (IC50: 2.90 μm), reflecting its weak anti‐thrombin activity.

Edoxaban prolongs PT and significantly and dose-dependently reduces thrombus formation at doses of 0.5, 2.5, and 12.5 mg/kg; po; once[1].

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PD and PK studies in rats and monkeys [1]
There was significant FXa inhibition activity in plasma (86% and 94% inhibition) in rats 0.5 h after oral administration of Edoxaban/DU‐176b (2.5 and 5 mg kg−1) (Fig. 4A), which was sustained for up to 4 h. In cynomolgus monkeys, DU‐176b also elicited a rapid onset of anti‐FXa activity, reaching a peak at 4 h (93%) and persisting 24 h (11%) after dosing (Fig. 4B). The area under the curve (AUC) of plasma concentration and maximum concentration (Cmax) after 1 mg kg−1 DU‐176b dosing were 852 ± 284 ng·h mL−1 and 175 ± 74 ng mL−1 (n = 6, mean ± standard deviation). Compared with DU‐176b, DX‐9065a had a lower anti‐FXa potency in both species (Fig. 4). AUC and Cmax in cynomolgus monkeys after the 1 mg kg−1 DX‐9065a dosing were 191 ± 104 ng·h mL−1 and 36.8 ± 20.5 ng mL−1 (n = 6).

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Antithrombotic effects of orally administered Edoxaban/DU‐176b [1]
Venous stasis thrombosis model in rats and rabbits Infusion of hypotonic saline and stasis of the inferior vena cava in rats led to the formation of thrombi weighing 4.38 ± 0.53 mg. Oral administration of DU‐176b (0.5, 2.5 and 12.5 mg kg−1) significantly and dose‐dependently reduced the thrombus formation (Fig. 5A) and prolonged PT (Fig. 5B). The plasma samples derived from DU‐176b‐treated rats inhibited exogenous FXa activity (Fig. 5C). In rabbits, DU‐176b also exerted a dose‐dependent antithrombotic effect (Fig. 5D), PT prolongation, and anti‐FXa activity in plasma (data not shown), significantly decreasing thrombi by 91% at 3 mg kg−1.

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Platinum wire‐induced venous thrombosis model [1]
Placement of a platinum wire in the rat vein induced formation of thrombi weighing 2.45 ± 0.38 mg on the surface of the wire. The thrombus formation was significantly reduced by Edoxaban/DU‐176b in a dose‐dependent manner (Fig. 6A). At a dose of 2.5 mg kg−1, DU‐176b reduced thrombus formation to 0.73 ± 0.21 mg. Similarly, FXa inhibition activity in plasma was significant and dose‐dependent (Fig. 6B).

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Effect on bleeding time [1]
Effect of Edoxaban/DU‐176b on tail bleeding time was not significantly different from control at 3 mg kg−1 (Table 3). At higher doses (10 and 30 mg kg−1), bleeding time was significantly prolonged (1.9‐fold) compared with the control.

Anti‐FXa activity of Edoxaban/DU‐176b [1]
To determine the inhibitory effect of DU‐176b on FXa activity, FXa was added to the mixture of DU‐176b or 5% dimethylsulfoxide (DMSO) control and a chromogenic substrate S‐2222 (250–1000 μm) in a reaction buffer (20 mm Tris–HCl, pH 7.4, 150 mm NaCl, 0.1% BSA). The final concentrations of FXa were as follows: human FXa (0.005 U mL−1, 0.7 nm), rabbit FXa (0.005 U mL−1, molarity unavailable), rat FXa (0.025 U mL−1, 10 nm) and cynomolgus monkey FXa (0.025 U mL−1, 3 nm). To measure amidolysis of S‐2222 by FXa, the absorbance at 405 nm was monitored with a microplate spectrophotometer SPECTRAmax 340 (Molecular Devices, Sunnyvale, CA, USA) at 30 °C for 10 min and the reaction velocity (mO.D./min) was obtained. The inhibition constant (Ki) values of DU‐176b were calculated by the Lineweaver–Burk plots and subsequent secondary plots.

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Inhibition of prothrombinase by Edoxaban/DU‐176b [1]
The inhibitory effect of DU‐176b on prothrombinase activity was examined using S‐2222 and the physiological substrate prothrombin, as described by Rezaie. Briefly, lipid vesicles were prepared by mixing of 1.2 mm phosphatidylcholine and 0.4 mm phosphatidylserine in chloroform, drying under vacuum, and resuspending in 9% sucrose. The suspension was sonicated and vesicles were extruded through filters of pore size 50–200 nm. Prothrombinase was formed by mixing human FXa (0.4 nm for S‐2222 and 0.2 pm for prothrombin), FVa (10 nm), CaCl2 (2.5 mm), and phosphatidylcholine/phosphatidylserine vesicles (25 μm) at 37 °C for 5 min. Amidolysis of S‐2222 (250–1000 μm) was measured as described for anti‐FXa activity of DU‐176b. Thrombin generation from prothrombin (7.8–250 nm) was measured as follows: the prothrombinase reaction proceeded for 3 min and was stopped by the addition of 10 mm EDTA. The activity of generated thrombin was measured by the amidolysis of its substrate S‐2238 and the concentration of thrombin was determined from a standard curve. The Ki values were calculated using the Lineweaver‐Burk plots and subsequent secondary plots.

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Specificity of serine protease inhibition of Edoxaban/DU‐176b [1]
The effects of DU‐176b on the following serine proteases (final concentrations) were examined: thrombin (0.03 U mL−1, 0.5 nm), FVIIa/sTF (2 nm/20 nm), FIXa (6.25 U mL−1, molarity unavailable), FXIa (0.25 nm), tPA (750 U mL−1, 20 nm), aPC (2.5 nm), trypsin (0.3 U mL−1, 1 nm), plasmin (0.004 U mL−1, 4 nm), and chymotrypsin (0.005 U mL−1, 2.5 nm). The enzymatic activities were assessed by the amidolysis of the following chromogenic substrates for correspondent protease: S‐2238 for thrombin, Spectrozyme fVIIa for FVIIa/sTF, Spectrozyme fIXa for FIXa, S‐2366 for FXIa and aPC, S‐2288 for tPA, S‐2251 for plasmin, S‐2222 for trypsin, and S‐2586 for chymotrypsin. The Ki values for these enzymes were determined as previously described.

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Anticoagulant activity in vitro [1]
The in vitro anticoagulant effects of Edoxaban/DU‐176b were studied. Clotting time (CT) in human, rat, cynomolgus monkey and rabbit plasma was measured using a microcoagulometer Amelung KC‐10A (MC Medical, Tokyo, Japan) and anticoagulant activity was expressed as the concentration of DU‐176b required to double CT (CT2), estimated by regression analysis from the dose‐response curves. Prothrombin time (PT) was measured by incubating plasma and DU‐176b (control; 4% DMSO/saline) for 1 min at 37 °C, followed by the addition of Thromboplastin C Plus (final concentration 0.25 U mL−1). Activated partial thromboplastin time (APTT) was measured by incubating plasma, DU‐176b and Platelin LS for 5 min at 37 °C, followed by the addition of CaCl2 (8.3 mm). Thrombin time (TT) was measured by incubating plasma and DU‐176b for 1 min at 37 °C, followed by the addition of human thrombin (4 U mL−1).

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Platelet aggregation [1]
Platelet‐rich plasma (PRP) was prepared from blood samples of healthy volunteers by centrifugation at 200 × g for 10 min at room temperature. To prepare washed platelets, PRP was then centrifuged at 600 × g for 10 min and the pellet was washed three times in Cor buffer (138 mm NaCl, 2.9 mm KCl, 10 mm Hepes‐NaOH, pH 7.3, 5.5 mm glucose, 12 mm NaHCO3) containing prostaglandin E1 (1 μm) and EDTA (10 mm). Washed platelets (2 × 108 platelets mL−1) were suspended in Cor buffer containing fibrinogen (1 mg mL−1) and CaCl2 (1 mm). EdoxabanDU‐176b was added to PRP or washed platelet suspension and incubated for 2 or 4 min at 37 °C. Platelet aggregation (>60%) was induced by the addition of collagen (0.8 μg mL−1), U46619 (0.7 μm) or ADP (5 μm) in PRP, and thrombin (0.08 U mL−1) in washed platelet suspension. Platelet aggregation was measured using an aggregometer PAM‐12C (MC Medical). Regression analysis was used to calculate the IC50 of DU‐176b.

Cell Viability Assay [1]
Cell Types: Human, rat, cynomolgus monkey and rabbit plasma; Human platelet
Tested Concentrations:
Incubation Duration: 1 and 5 minutes
Experimental Results: Antithrombin.

Animal/Disease Models: Male Slc: Wistar rats (210-240 g); Male New Zealand White rabbits(2.5-3.5 kg) (Both are venous stasis thrombosis model)[1].
Doses: 0.5, 2.5 and 12.5 mg/kg
Route of Administration: Oral administration; once
Experimental Results: Inhibited exogenous FXa activity. Antithrombotic.

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PD and PK studies of Edoxaban/DU‐176b after oral administration to rats and cynomolgus monkeys [1]
DU‐176b, DX‐9065a or the 0.5% methylcellulose vehicle were administered orally to fasted animals by gavage, and citrated blood samples were collected at 0.5, 1, 2 and 4 h in rats (n = 4 per dose group), and 0.5, 1, 2, 4, 8 and 24 h in cynomolgus monkeys (n = 6 per dose group) after administration. To measure FXa inhibition activity in plasma, a plasma sample (5 μL) was added to the reaction mixture of human FXa (0.01 U mL−1, 1.4 nm) and S‐2222 (300 μm). Amidolysis of S‐2222 was measured as described. The plasma concentrations of DU‐176b and DX‐9065a were measured by high‐performance liquid chromatography with tandem mass spectrometric detection.

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Antithrombotic effects of orally administered Edoxaban/DU‐176b [1]
Venous stasis thrombosis model in rats DU‐176b (0.5–12.5 mg kg−1) or 0.5% methylcellulose was orally administered to fasted rats (n = 8 per dose group). Venous thrombosis was induced 30 min after DU‐176b administration according to the method by Hladovec while the animals were anesthetized with thiopental sodium (100 mg kg−1, i.p.). Briefly, hypotonic NaCl solution (0.225%) was injected into the femoral vein (5 mL kg−1 min−1 for 2 min), and the inferior vena cava was ligated just below the left renal vein. Ten minutes later, the vena cava was ligated again 1.5 cm below the first ligature. The resulting thrombus was removed 1 h after the second ligation and its wet weight was measured. Blood samples were collected 29 min after DU‐176b dosing to measure PT and plasma FXa inhibition activity.

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Venous stasis thrombosis model in rabbits [1]
DU‐176b/Edoxaban (0.3–3 mg kg−1) or 0.5% methylcellulose was administered orally to fasted rabbits (n = 8 per dose group). The rabbits were anesthetized with urethane (2 g kg−1, i.p.) and venous thrombosis was induced 45 min after DU‐176b administration according to the method by Wessler et al. with some modifications. Recombinant human TF (0.05 μg 2‐mL−1 kg−1 for 30 s) was injected into the auricular vein, and 15 s later blood stasis was made in a 2‐cm segment of the jugular vein by a pair of ligations. The resulting thrombus was removed after 30 min, and its wet weight was measured.

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Platinum wire‐induced venous thrombosis model in rats [1]
Thrombus was induced by the insertion of a platinum wire (2 cm long) into the inferior vena cava of rats (n = 8 per dose group) just caudal to the left renal vein 30 min after oral administration of Edoxaban/DU‐176b (0.1–2.5 mg kg−1) or 0.5% methylcellulose according to the method of Lavelle and Iomhair. The resulting thrombus was fixed 1 h later with 1% glutaraldehyde. The wet weight of the thrombus was measured and blood samples were collected 29 min after DU‐176b dosing to measure plasma FXa inhibition activity.

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Effect of Edoxaban/DU‐176b on bleeding time [1]
Hemorrhage was induced 30 min following oral administration of DU‐176b (3–30 mg kg−1) or 0.5% methylcellulose and bleeding time was measured in a rat tail bleeding model. Briefly, an incision (1 mm deep) was made 4 cm from the tip of the tail. Blood was blotted every 15 s on filter papers, and bleeding time was defined as the time from the incision to the first arrest of bleeding. The maximum observation period was 30 min and longer bleeding time was assigned a value of 30 min.

[1]. DU-176b, a potent and orally active factor Xa inhibitor: in vitro and in vivo pharmacological profiles. J Thromb Haemost. 2008 Sep;6(9):1542-9.

[2]. The Effects of the Antiplatelet Agents, Aspirin and Naproxen, on Pharmacokinetics and Pharmacodynamics of the Anticoagulant Edoxaban, a Direct Factor Xa Inhibitor. J Cardiovasc Pharmacol. 2013 Apr 23.

Therapeutic Uses
Xa Factor Inhibitors
/Clinical Trials/ ClinicalTrials.gov is a registry and results database that lists human clinical studies funded by public and private institutions worldwide. The website is maintained by the National Library of Medicine (NLM) and the National Institutes of Health (NIH). Each record on ClinicalTrials.gov includes a summary of the study protocol, including: the disease or condition; the intervention (e.g., the medical product, behavior, or procedure under investigation); the title, description, and design of the study; participation requirements (eligibility criteria); the location of the study; contact information for the study location; and links to relevant information from other health websites, such as the NLM's MedlinePlus (for providing patient health information) and PubMed (for providing citations and abstracts of academic articles in the medical field). Edoxaban is listed in the database.
Savaysa is indicated for reducing the risk of stroke and systemic embolism (SE) in patients with nonvalvular atrial fibrillation (NVAF). /US Product Label Includes/
Savaysa is indicated for the treatment of deep vein thrombosis (DVT) and pulmonary embolism (PE) 5 to 10 days after initial parenteral anticoagulation therapy. /US Product Label Includes/
For more complete data on the therapeutic uses of edoxaban (of 7), please visit the HSDB record page.

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Drug Warning
/Black Box Warning/ Reduced efficacy in patients with nonvalvular atrial fibrillation with creatinine clearance (CrCL) > 95 mL/min. Savaysa should not be used in patients with CrCL > 95 mL/min. In the ENGAGE AF-TIMI 48 study, patients with nonvalvular atrial fibrillation and creatinine clearance (CrCL) > 95 mL/min had an increased incidence of ischemic stroke after once-daily administration of 60 mg savaysa compared to patients receiving warfarin. For these patients, an alternative anticoagulant should be used.
/Black Box Warning/ Premature discontinuation of savaysa increases the risk of ischemic events. In the absence of adequate alternative anticoagulation therapy, premature discontinuation of any oral anticoagulant increases the risk of ischemic events. If Savaysa is discontinued for pathological bleeding or for reasons other than completion of the course of treatment, consider using an alternative anticoagulant as described in the transition guidelines.
/Warning/ Spinal/Epidural Hematoma. Epidural or spinal hematomas may occur in patients receiving Savaysa and undergoing spinal anesthesia or lumbar puncture. These hematomas can lead to long-term or permanent paralysis. These risks should be considered when scheduling patients for spinal surgery. Factors that may increase the risk of epidural or spinal hematoma in such patients include: use of an indwelling epidural catheter; concurrent use of other medications that affect hemostasis, such as nonsteroidal anti-inflammatory drugs (NSAIDs), platelet inhibitors, or other anticoagulants; a history of traumatic or recurrent epidural or spinal punctures; a history of spinal deformity or spinal surgery; the optimal time interval between Savaysa administration and spinal surgery is not well understood. Patients should be closely monitored for signs and symptoms of neurological dysfunction. If neurological impairment is detected, treatment must be initiated immediately. For patients currently receiving or about to receive anticoagulation therapy, the risks and benefits should be weighed before initiating spinal intervention. The safety and efficacy of edoxaban in patients who have undergone mechanical heart valve replacement or have moderate to severe mitral stenosis have not been evaluated; its use is not recommended in these patients. For more drug warnings about edoxaban (full version) (18 in total), please visit the HSDB record page. Pharmacodynamics: Edoxaban can prolong coagulation time test results, such as activated partial thromboplastin time (aPTT), prothrombin time (PT), and international normalized ratio (INR). Edoxaban is a monocarboxylic acid amide (in its tosylate monohydrate form) used to treat deep vein thrombosis and pulmonary embolism. It has anticoagulant, EC 3.4.21.6 (coagulation factor Xa) inhibitor, and platelet aggregation inhibitory effects. It is a monocarboxylic acid amide, chloropyridine, thiazopyridine, and tertiary amine compound. It is the conjugate base of edoxaban (1+). Edoxaban belongs to the class of novel oral anticoagulants (NOACs) and is a rapid-acting, oral, selective factor Xa inhibitor. Edoxaban inhibits the stepwise amplification of factor Xa, a key protein in the coagulation cascade, preventing thrombus formation. This product is indicated for reducing the risk of stroke and systemic embolism (SE) in patients with nonvalvular atrial fibrillation (NVAF), and for treating deep vein thrombosis (DVT) and pulmonary embolism (PE) after 5-10 days of initial parenteral anticoagulation therapy. Traditionally, warfarin (a vitamin K antagonist) has been used to prevent stroke in these patients, but its effective use has been limited due to its slow onset of action, narrow therapeutic window, need for regular monitoring and INR testing, and various drug-drug and drug-food interactions. This has spurred considerable interest in novel antithrombotic drugs such as dabigatran, apixaban, and rivaroxaban. In addition to once-daily dosing, edoxaban's advantages over warfarin include a significantly reduced risk of hemorrhagic stroke and gastrointestinal bleeding, as well as improved patient adherence, which is crucial for many patients requiring lifelong treatment. Edoxaban is a factor Xa inhibitor. Its mechanism of action is as a factor Xa inhibitor. Edoxaban is an oral small-molecule factor Xa inhibitor used as an anticoagulant to reduce the risk of venous thrombosis, systemic embolism, and stroke in patients with atrial fibrillation, and is used to treat deep vein thrombosis and pulmonary embolism. Edoxaban is associated with a low incidence of elevated serum transaminases during treatment and rare cases of clinically significant acute liver injury. Edoxaban is an orally effective inhibitor of coagulation factor Xa (activating factor X) with anticoagulant activity. Edoxaban is administered in the form of edoxaban tosylate. The elimination half-life of this drug is 9-11 hours, and it is primarily excreted via the kidneys. Edoxaban is a small molecule drug, with its clinical trial phase reaching up to Phase IV (covering all indications). It was first approved in 2015 and currently has 6 approved indications and 15 investigational indications. This drug has been placed on the black box warning list by the U.S. Food and Drug Administration (FDA). Background: Factor Xa (FXa) is a key serine protease that converts prothrombin to thrombin in the coagulation cascade and is a promising target enzyme for the prevention and treatment of thromboembolic diseases. Edoxaban/DU-176b is a novel antithrombotic drug that directly inhibits FXa activity. Objective: To evaluate the in vitro pharmacological properties and in vivo effects of DU-176b in animal models of thrombosis and hemorrhage. Methods: In vitro FXa inhibitory activity, specificity, and anticoagulant activity were assessed. Oral absorption was studied in rats and cynomolgus monkeys. In vivo effects were investigated in rat and rabbit models of venous thrombosis and tail hemorrhage. Results: DU-176b/edoxaban showed a Ki value of 0.561 nM for free FXa and 2.98 nM for prothrombinase, exhibiting over 10,000-fold selectivity for FXa. In human plasma, DU-176b concentrations of 0.256 μM and 0.508 μM both doubled prothrombin time and activated partial thromboplastin time. DU-176b did not affect ADP, collagen, or U46619-induced platelet aggregation. DU-176b was well absorbed in rats and monkeys, and its anti-Xa activity was stronger after oral administration, with plasma drug concentrations higher than those of the original FXa inhibitor DX-9065a. In vivo experiments showed that DU-176b inhibited thrombosis in rat and rabbit thrombosis models in a dose-dependent manner, although bleeding time in rats was not significantly prolonged at antithrombotic doses. Conclusion: Compared with the parent drug DX-9065a, DU-176b/edoxaban is a more potent and selective FXa inhibitor with higher oral bioavailability. DU-176b is a promising new anticoagulant for the prevention and treatment of thromboembolic diseases. [1] In summary, edoxaban/DU-176b is a potent and highly selective direct FXa inhibitor with significantly improved potency, selectivity and oral bioavailability compared with DX-9065a. This study shows that DU-176b has the potential as an oral antithrombotic drug and is a promising new anticoagulant for the prevention and treatment of thromboembolic diseases. [1]

C16H22CLN5O3

367.83

480452-37-7

White to off-white solid powder

1.34±0.1 g/cm3 (20 ºC 760 Torr)

0

ClC1=CN=C(C=C1)NC(C(N[C@H]1CC[C@H](C(N(C)C)=O)C[C@H]1N)=O)=O

480452-37-7; EthanediaMide iMpurity F; Edoxaban impurity 6; N1-((1S,2R,4S)-2-amino-4-(dimethylcarbamoyl)cyclohexyl)-N2-(5-chloropyridin-2-yl)oxalamide; SCHEMBL1253410; IZABLUDXAWYTNU-WCQGTBRESA-N; N-[(1S,2R,4S)-2-amino-4-(dimethylcarbamoyl)cyclohexyl]-N'-(5-chloropyridin-2-yl)oxamide; N1-((1S,2R,4S)-2-Amino-4-(dimethylcarbamoyl)cyclohexyl)-N2-(5-chloropyridin-2-yl)oxalamide (Edoxaban Impurity);

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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