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.

---

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.

---

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.

---

View More

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.

---

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).

---

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.

---

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.

---

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)].

---

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).

---

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.

---

View More

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.

---

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.

---

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).

---

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
Rivaroxaban is rapidly absorbed after oral administration, reaching peak plasma concentrations within 2–4 hours. The bioavailability of a 10 mg dose is >80%. However, bioavailability is lower when taken on an empty stomach at doses of 15–20 mg; therefore, it should be taken with food. Approximately two-thirds of rivaroxaban is excreted in the urine (actively secreted by the renal tubules, of which approximately 36% is the unchanged drug and 30% is inactive metabolites). The remaining one-third is excreted in the feces, of which 7% is the unchanged drug and 21% is inactive metabolites. The steady-state volume of distribution (Vd) is 50 L. Systemic clearance is approximately 10 L/h; therefore, rivaroxaban is considered a low-clearance drug. Renal clearance is approximately 3–4 L/h. After oral administration, approximately one-third of the absorbed dose is excreted unchanged in the urine, and the remaining two-thirds are excreted as inactive metabolites in the urine and feces. In a phase I study, following administration of (14)C-rivaroxaban, 66% of the radioactive dose was recovered in the urine (36% of which was unchanged drug) and 28% in the feces (7% of which was unchanged drug). The unchanged drug was primarily excreted in the urine via active tubular secretion, with a small amount excreted via glomerular filtration (approximately 5:1). Rivaroxaban is a substrate of the efflux transporters P-gp and ABCG2 (also known as Bcrp). The affinity of rivaroxaban for influx transporters is not well understood. Rivaroxaban binds to approximately 92% to 95% of plasma proteins in human plasma, with albumin being the primary binding protein. The steady-state volume of distribution in healthy subjects is approximately 50 liters. The absorption of rivaroxaban depends on the site of release in the gastrointestinal tract. When rivaroxaban granules are released in the proximal small intestine, their AUC and Cmax are reduced by 29% and 56%, respectively, compared to tablets. Drug exposure is further reduced when the drug is released in the distal small intestine or ascending colon. Administration to the distal stomach should be avoided as this leads to reduced absorption and associated drug exposure. Rivaroxaban's absolute bioavailability is dose-related. For a 10 mg dose, bioavailability is estimated to be 80% to 100%, unaffected by food. Xarelto 10 mg tablets can be taken with or without food. The absolute bioavailability of a 20 mg dose taken with food is approximately 66%. Taking it with food increases the bioavailability of the 20 mg dose (mean AUC and Cmax increase by 39% and 76%, respectively). Xarelto 15 mg and 20 mg tablets should be taken with food. For more complete data on absorption, distribution, and excretion of rivaroxaban (8 metabolites), please visit the HSDB record page. Approximately two-thirds of the dose is metabolized. Rivaroxaban is primarily metabolized via CYP3A4, CYP3A5, CYP2J2, and CYP-independent mechanisms. Rivaroxaban is mainly degraded by oxidative degradation and hydrolysis of cytochrome P-450 (CYP) isoenzymes 3A4/5 and 2J2; the metabolites are subsequently excreted via the kidneys and feces/bile. No major circulating metabolites have been detected in plasma. Biological Half-Life: The terminal half-life in adults is 5–9 hours, and in older adults it is 11–13 hours. The terminal elimination half-life in older adults is 11–13 hours. The terminal elimination half-life of rivaroxaban in healthy subjects aged 20–45 years is 5–9 hours.

Hepatotoxicity
In patients taking rivaroxaban long-term, 1.5% to 3% experience moderate ALT elevation (more than 3 times the upper limit of normal), with an overall incidence slightly lower than low molecular weight heparin and similar to warfarin. Several cases of ALT elevation with jaundice were observed in large pre-marketing clinical trials of rivaroxaban, but little detail was provided at the time, and it was unclear whether liver injury was clinically manifested. These cases were apparently mild and self-limiting, recovering completely upon discontinuation of the drug. Since rivaroxaban's approval and widespread use, several cases of acute liver injury with jaundice have been identified. The clinical presentations of these cases vary considerably. Most cases develop within 1 to 8 weeks of starting rivaroxaban, presenting with jaundice, fatigue, and elevated hepatocellular serum enzymes. Some patients develop cholestasis or mixed liver injury. Immune hypersensitivity features and autoimmune markers are atypical, but at least one case presented with rash and fever, suggesting a drug reaction accompanied by eosinophilia and systemic symptoms (DRESS). One case of acute liver necrosis and death caused by rivaroxaban has been reported, but the more likely cause was ischemic hepatitis due to severe heart failure. All other reported cases of liver injury caused by rivaroxaban recovered after discontinuation of rivaroxaban, usually rapidly within 2 to 4 weeks. Acute liver injury hospitalizations account for approximately 1 in 2200 in large healthcare databases, but it is uncertain whether all cases in these databases are caused by rivaroxaban. Probability score: A (Clinically evident cause of liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation Multiple case reports and a comprehensive pharmacokinetic analysis consistently indicate that when mothers take 15 to 30 mg of rivaroxaban daily, the drug concentration in breast milk is very low, far below the dose required for infant anticoagulation (<2%). Rivaroxaban was undetectable in the plasma of two breastfed infants. This is not a reason to discontinue breastfeeding if the mother needs to take rivaroxaban.
◉ Effects on Breastfed Infants
A 38-year-old woman with antiphospholipid syndrome started taking rivaroxaban 15 mg (0.19 mg/kg) once daily, 5 days postpartum, for the prevention of deep vein thrombosis. She partially breastfed her infant (at least 50%). No significant bleeding was observed in the infant at 1-month and 3-month checkups, and the infant was developing normally at 18 months.
Two mothers started taking rivaroxaban 15 mg (0.22 and 0.25 mg/kg) once daily, 3 days postpartum, for the prevention of deep vein thrombosis. At 3 months postpartum, the infants continued breastfeeding (the extent of breastfeeding was not specified), and no health problems or bleeding events occurred.
◉ Effects on Lactation and Breast Milk
As of the revision date, no relevant published information was found.
Plasma protein binding is approximately 92% to 95%.

Drug Interactions

Patients with renal impairment who are concurrently taking full-dose rivaroxaban and P-gp in combination with a weak or intermediate-acting CYP3A4 inhibitor (e.g., amiodarone, diltiazem, verapamil, quinidine, ranolazine, dronedarone, felodipine, erythromycin, and azithromycin) may have higher drug exposure than patients with normal renal function who are not using inhibitors, because both clearance pathways of rivaroxaban are affected. For patients with a creatinine clearance (CrCl) of 15 to 50 mL/min who are concurrently receiving a P-gp inhibitor and a weak or intermediate-acting CYP3A4 inhibitor, rivaroxaban (Xarelto) should only be used if the potential benefit outweighs the potential risk.
Concurrent administration of enoxaparin and rivaroxaban in a single dose may have an additive effect on factor Xa activity.
Concomitant administration of warfarin and rivaroxaban in a single dose can have an additive effect on factor Xa inhibition and prothrombin time (PT). In efficacy trials, concomitant aspirin use has been identified as an independent risk factor for major bleeding. Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to increase the risk of bleeding, and this risk may be increased when NSAIDs are used concomitantly with rivaroxaban. Concomitant use of the platelet aggregation inhibitor clopidogrel with rivaroxaban can lead to prolonged bleeding time in some subjects. Rivaroxaban should be avoided concomitantly with other anticoagulants unless the benefits outweigh the risks, as this increases the risk of bleeding. If a patient is taking aspirin, other platelet aggregation inhibitors, or NSAIDs, any signs or symptoms of bleeding should be evaluated immediately.
Population pharmacokinetic analysis results from drug interaction studies and clinical trials indicate that concomitant use of rivaroxaban with P-gp and potent CYP3A4 inducers (e.g., rifampin, phenytoin sodium) can reduce rivaroxaban exposure by up to 50%. Similar reductions in pharmacodynamic effects have also been observed. This reduction in rivaroxaban exposure may decrease therapeutic efficacy. Avoid concomitant use of xareltoxin with P-gp and potent CYP3A4 inducers (e.g., carbamazepine, phenytoin sodium, rifampin, St. John's wort).
No precautions are necessary when concomitant use with P-gp and CYP3A4 inhibitors if data indicate that changes in exposure are unlikely to affect bleeding risk (e.g., clarithromycin, erythromycin). Avoid concomitant use of xareltoxin with P-gp and potent CYP3A4 inhibitors.
For more complete data on drug interactions of rivaroxaban (10 in 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

Anticoagulants
Xarelto is indicated for the treatment of deep vein thrombosis (DVT). /US Product Label/
Xarelto is indicated for the reduction of the risk of stroke and systemic embolism in patients with nonvalvular atrial fibrillation. Currently, there are limited data on the relative efficacy of Xarelto and warfarin in reducing the risk of stroke and systemic embolism when well controlled with warfarin. /US Product Label/
Xarelto is indicated for the prevention of deep vein thrombosis in patients undergoing knee or hip replacement surgery, which can lead to pulmonary embolism. /Included in US Product Label/
For more complete data on the therapeutic uses of rivaroxaban (out of 7), please visit the HSDB record page.

---

Drug Warnings
/Black Box 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 Xarelto is discontinued for pathological bleeding or for reasons other than completion of the course of treatment, consider using an alternative anticoagulant. /Warning (Black Box)/ Warning: Spinal/Epidural Hematoma. Patients who have received Xareltox and undergone spinal anesthesia or spinal puncture have a history of epidural or spinal hematoma. 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 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; and a history of spinal deformities or spinal surgery. Patients should be closely monitored for signs and symptoms of neurological dysfunction. If neurological impairment is detected, immediate treatment is necessary. For patients receiving or about to receive anticoagulation therapy to prevent thrombosis, the risks and benefits should be weighed before spinal intervention. Rivaroxaban increases the risk of bleeding and can lead to serious or fatal bleeding. In clinical trials, the most common adverse reaction of rivaroxaban was bleeding complications. Rivaroxaban should be avoided in patients with moderate (Child-Pugh B) or severe (Child-Pugh C) hepatic impairment, or any liver disease with coagulation disorders; these patients may have increased systemic exposure and bleeding risk. For more complete (13) drug warnings for rivaroxaban, please visit the HSDB record page.

---

Pharmacodynamics
Rivaroxaban is an anticoagulant that binds directly to factor Xa. It then effectively blocks the amplification of the coagulation cascade, thereby preventing thrombosis. Rivaroxaban is a unique anticoagulant for two reasons. First, it exerts its anticoagulant effect without involving antithrombin III (ATIII). Second, rivaroxaban is an oral medication, while widely used unfractionated heparin and low molecular weight heparin are only for parenteral administration. Although activated partial thromboplastin time (aPTT) and HepTest (a test for detecting low molecular weight heparin) may increase with dose, neither of these tests is recommended for assessing the pharmacodynamic effects of rivaroxaban. While rivaroxaban may affect anti-Xa activity and its inhibitory effects, monitoring of these parameters is not recommended.

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.

---

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.

---

View More

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

---

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