Factor Xa

The first novel anticoagulant that targets factor Xa specifically is fondaparinux sodium. The IC50 value (anti-Xa IU/ml) for fondaparinux is 0.59±0.05 for activated monocytes (ac-M) and 0.17±0.03 for MMPs (monocyte-derived microparticles) [2].

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Effect of Fondaparinux, low molecular weight heparin and unfractionated heparin on thrombin generation supported by activated monocytes [2]
Values obtained at baseline were 2.1 ± 0.1 min for lag time, 181 ± 6 nmol/l for peak, 1624 ± 84 nmol/l min for ETP and 59 ± 4 nmol/l/min for rate index. Fondaparinux, enoxaparin and UFH inhibited ac-M-supported thrombin generation in a concentration-dependent manner (Fig. 1). Except for the lag time, the inhibitory effect of UFH was superior to enoxaparin, which was superior to fondaparinux, from 0.3 to 1.0 anti-Xa IU/ml. Whereas comparable inhibitory effect of fondaparinux and enoxaparin on peak and ETP was noted at the lowest anti-Xa concentration (0.1 IU/ml), the inhibitory effect on rate index of fondaparinux was more pronounced than that of enoxaparin at 0.1 IU/ml anti-Xa concentration.

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Calculation of IC50 [2]
The inhibitory effect on thrombin generation was evaluated by calculating the inhibitory concentration (IC)50, which was defined for peak, ETP and rate index as the drug concentration yielding 50% reduction, and for lag time as the drug concentration resulting in doubling it. UFH was the only drug for which IC50 was calculated for all thrombin generation parameters in the two models. In contrast, enoxaparin IC50 values were calculated for ETP, peak and rate index, but not for lag time, in the two models. Furthermore, IC50 for Fondaparinux was reached only for rate index in the two models and for peak and ETP only in the MMP model. Rate index was the only parameter for which IC50 was reached in both models for the three drugs; UFH was the most effective, followed by enoxaparin and fondaparinux (Table 1).

The pharmacokinetics of fondaparinux sodium are linear and dose-dependent, resulting in a very predictable response. With a quick start of action, a half-life of 14 to 16 hours, and continuous antithrombotic activity for 24 hours, fondaparinux sodium is 100% bioavailable. The medication has no effect on platelet function or aggregation, prothrombin time, or activated partial thromboplastin time [1].

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Compared with the ex-vivo anti-Xa levels reported in Organization to Assess Strategies in Acute Ischemic Syndromes (OASIS)-5 substudy, our results demonstrate that anti-Xa values obtained in patients are either similar or even higher than those required in our in-vitro models used to reach rate index IC50. Fondaparinux anti-Xa level of 0.52 (±0.22) IU/ml in OASIS-5 study was sufficient to inhibit rate index in both in-vitro models tested. For enoxaparin, the anti-Xa level measured in OASIS-5 substudy patients was 1.2 (±0.45) IU/ml, which was markedly more superior than anti-Xa concentrations required to inhibit rate index in the two models of thrombin generation tested here (ac-M IC50: 0.27 ± 0.03 IU/ml, and MMPs IC50 0.19 ± 0.02 IU/ml).

The OASIS-5 substudy reported less inhibition of ETP in Fondaparinux-treated patients versus enoxaparin. Our in-vitro ETP results are in agreement with these data, taking into consideration that the Oasis-5 substudy ETP was performed in cell-free assay. Fondaparinux yielded lower inhibition than enoxaparin in both models, with anti-Xa concentrations ranging from 0.3 to 1.0 IU/ml. Fondaparinux ETP IC50 values were not always reached despite increased anti-Xa levels (fondaparinux tested at concentrations ranging from 0.1 to 1.0 anti-Xa IU/ml), and its ETP IC50 was reached only in the MMPs model. In contrast, enoxaparin reached ETP IC50 in the two in-vitro models tested here. As for rate index, the levels of anticoagulation in OASIS-5 enoxaparin-treated patients are much more superior to those required to inhibit ETP in our cell models.

Fondaparinux at 2.5 mg daily (corresponding to anti-Xa of 0.52 ± 0.22 IU/ml) in the Oasis-5 study, showed an optimal efficacy safety in ACS patients. This anti-Xa level for fondaparinux corresponded to the rate index IC50 of the ac-M model and to the ETP IC50 value of the MMP model. At this anti-Xa concentration, rate index was inhibited in the MMP model and ETP IC50 was not attained in ac-M model. For enoxaparin, rate index IC50 in ac-M model was similar to ETP IC50 in MMP model and was closely equal to 0.3 IU/ml. At this anti-Xa level, rate index in MMP model was largely inhibited and ETP IC50 in ac-M was not reached. Given this, it is assumed that an anti-Xa level of about 0.3 IU/ml for enoxaparin is sufficient in ACS for an optimal efficacy-safety profile. It should be noted that, of the three agents analysed here, fondaparinux is the only agent for which the daily dose has been rationally determined, as evidenced by the use of 2.5 mg dose on the basis of previous dose-finding studies [2].

Monocyte activation [2]
Purified monocytes were washed in RPMI-1640 medium and then adjusted to 1.5 106 cells/ml in RPMI-1640 containing 5% (v/v) heat-inactivated foetal calf serum, 2 mmol/l L-glutamine and 100 ng/ml LPS (Escherichia coli serotype O55:B5, Sigma). Following incubation of the monocytes for 5 h at 37°C in a 5% CO2 humidified atmosphere, supernatants were removed by centrifugation for 5 min at 400g. The ac-M-containing pellet was resuspended in 150 μl phosphate-buffered saline.

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Monocyte-derived microparticles preparation and quantification [2]
Monocytes were incubated for 18 h, as described above, and supernatants were collected by centrifugation for 5 min at 2200g. MMPs were recovered following an additional centrifugation at 17000g for 30 min and quantified by flow cytometry [5]. MMPs (100 μl) were incubated for 15 min in the dark with 1 μl annexin V-FITC, according to manufacturer's recommendations, followed by the addition of 400 μl annexin V-binding buffer and 100 μl flow-count Fluorospheres. Fluorescence was acquired for 60 s on Epics XL-MCL flow cytometer, using System 2 software. MMPs were quantified using the flow-count Fluorospheres and expressed as MMPs/μl.

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Fluorogenic measurement of thrombin generation [2]
Thrombin generation test was performed according to the assay described by Hemker and modified by Poitevin; ac-Ms and MMPs being defined as cell models. PPP used for all experimental conditions was systematically supplemented with aprotinin at 200 kallikrein inhibitory units (KUI)/ml final concentration. PPP was spiked with increasing concentrations of Fondaparinux, LMWH or UFH; final concentrations being 0.0, 0.1, 0.3, 0.6 and 1.0 anti-FXa IU/ml. We previously optimized the concentrations of ac-Ms and MMPs included in the assay. Thus, 20 μl of ac-M (0.2 × 106 ac-M per well) or of MMPs (160 000 MMPs per well) was added to 80 μl of PPP. Results were expressed as percentage, taking the baseline measurements as 100%.

Fluorometric determination of thrombin generation was done on Fluoroskan Ascent plate reader, following addition of the fluorogenic substrate, Z-Gly-Gly-Arg-AMC; Thrombinoscope software used for calculating thrombin generation. Four parameters of the thrombin generation curve were analysed: lag time (min), thrombin peak (peak, nmol/l), endogenous thrombin potential (ETP, nmol/l min), rate index of propagation phase defined by the formula: peak/(time to peak – lag-time) (rate index, nmol/l/min).

Platelet-poor plasma preparation [2]
Venous blood samples were obtained from five healthy volunteers (mean age 25 ± 2 years). Volunteers were laboratory staff members and did not receive any medication for the last 2 weeks. Informed consents were obtained from all of them. Blood was withdrawn by antecubital venipuncture into Monovette tubes (0.106 mol/l citrate). A three-step centrifugation procedure including 10 min at 190g, 10 min at 1750g and 30 min at 13 000g was used. Platelet-poor plasma (PPP) supernatants were pooled, stored at −80°C and thawed immediately before use.

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Monocyte purification [2]
Cytapheresis material was obtained from six healthy volunteers who were admitted for platelet donation in the blood transfusion unit of CHU Robert Debré. Informed consent was obtained from the participants. Monocytes were purified from cytapheresis residues by elutriation, as previously described. Monocyte purity was assessed by CD14 staining of isolated cells (>95% CD14-positive). Cell viability (>98%) was determined by the trypan blue exclusion principle. Purified monocytes were used for preparing ac-Ms and MMPs.

This article describes the pharmacology and mechanism of action of fondaparinux. Fondaparinux is the first novel anticoagulant that selectively targets factor Xa. It has linear dose-dependent pharmacokinetic characteristics, so its efficacy is highly predictable. It has 100% bioavailability, rapid onset of action, and a half-life of 14 to 16 hours, providing sustained antithrombotic effects for up to 24 hours. It does not affect prothrombin time or activated partial thromboplastin time, nor does it affect platelet function or aggregation. Studies in patients diagnosed with heparin-induced thrombocytopenia showed no cross-reactivity with in vitro heparin antibodies. Fondaparinux appears to meet the criteria for an ideal antithrombotic drug: efficacy comparable to or better than existing drugs, low risk of bleeding, no need for laboratory monitoring, and once-daily administration. [1]

Hepatotoxicity
The incidence of elevated serum transaminases during fondaparinux treatment is low, with 1% to 3% of patients experiencing transaminase levels exceeding three times the upper limit of normal, lower than heparin (approximately 8%) or low molecular weight heparin (4% to 12%), but higher than placebo (
Probability score: E (unlikely to cause clinically significant liver injury)).
Effects during pregnancy and lactation
◉ Overview of use during lactation
Fondaparinux is considered acceptable for use during lactation.
◉ Effects on breastfed infants
No relevant published information found as of the revision date.
◉ Effects on lactation and breast milk
No relevant published information found as of the revision date.
Interactions
To investigate in vitro metabolism, this study aimed to determine the metabolism of the antithrombotic drug fondaparinux in mammalian liver components and to assess its potential inhibitory effects on the metabolism of other drugs mediated by human cytochrome P450 (CYP). The metabolism of fondaparinux sodium was assessed by incubating it with post-mitochondrial liver fractions from rats, rabbits, monkeys, or humans (three subjects). Human liver microsomal formulations and NADPH-generating systems were incubated with phenacetin, coumarin, tolbutamide, S-mphenytoin, ibuprofen, chlorzoxazone, or nifedipine. These drugs are selectively metabolized by CYP isoenzymes CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, or CYP3A4, respectively. The experiments aimed to determine the apparent Ki (inhibition constant) value of fondaparinux sodium. The inhibitory effect of fondaparinux sodium on each CYP isoenzyme was investigated by varying the concentrations of fondaparinux sodium and selective substrates. Each experiment included a control reaction mixture containing an isoenzyme selective inhibitor. After incubation, the mixtures were analyzed by LC-MS/MS or fluorescence detection. All liver fractions exhibited enzymatic activity, as evidenced by the degradation of [(14)C]testosterone. Fondaparinux metabolism was not detected in the postmitochondrial fraction of the liver. Since the oxidative metabolism of isoenzyme-selective CYP substrates was not significantly inhibited in the mixed microsomal reaction mixture, the apparent Ki values of fondaparinux for CYP isoenzymes could not be determined. In the presence of selective CYP inhibitors, the metabolism of each substrate was significantly reduced, confirming the inhibitory effect observed in these assays. The lack of fondaparinux metabolism in mammalian livers is consistent with findings from animal and human studies. CYP isoenzymes are generally involved in drug metabolism, suggesting that clinical treatment with fondaparinux is unlikely to interfere with the pharmacokinetics and metabolism of many other drugs associated with CYP inhibition. PMID:12383041.. Two independent studies evaluated potential interactions between fondaparinux and aspirin (acetylsalicylic acid) or piroxicam in healthy volunteers under steady-state conditions. In the first study, the effects of a single 975 mg aspirin dose were first evaluated, followed by evaluation of a single aspirin dose or placebo dose on day four of an 8-day subcutaneous treatment regimen of fondaparinux sodium (10 mg, once daily). The second study was a phase III crossover, double-blind, randomized controlled trial investigating three regimens: fondaparinux sodium 10 mg + placebo, fondaparinux sodium 10 mg + piroxicam 20 mg, or placebo + piroxicam 20 mg. …No interactions were found in any of these regimens. Neither aspirin nor piroxicam affected the pharmacokinetics of fondaparinux sodium at steady state. Two hours after administration, the prolongation of bleeding time was significantly greater with aspirin alone or with aspirin in combination with fondaparinux sodium than with fondaparinux sodium alone (p = 0.003 and p = 0.004, respectively). There was no significant difference in the effect of aspirin alone or with fondaparinux sodium in combination on bleeding time. Collagen-induced platelet aggregation was slightly decreased after piroxicam alone or in combination with fondaparinux. The effect on activated partial thromboplastin time (aPTT) was minimal; changes in aPTT were similar when fondaparinux was administered alone or in combination with aspirin or piroxicam. No serious adverse events were reported. PMID: 12383043. This study evaluated potential pharmacokinetic and pharmacodynamic interactions between fondaparinux and digoxin in healthy men at home. Volunteers. In a phase I randomized crossover study, 24 volunteers (n = 24) received two phases of treatment. Phase I consisted of once-daily subcutaneous injections of 10 mg fondaparinux for 7 days; Phase II consisted of oral administration of 0.25 mg digoxin for 7 days, followed by 10 mg fondaparinux for 7 days. A 12-day washout period was provided between the two phases. ...The pharmacokinetic characteristics of digoxin and fondaparinux were not affected by the combination therapy. Bioequivalence was concluded based on the 90% confidence interval of the adjusted geometric mean ratio of peak concentration, area under the concentration-time curve, and cumulative urinary excretion (2×2 comparisons) within a reference range of 0.80 to 1.25. No clinically significant fluctuations were observed in vital signs and electrocardiographic parameters. The combination of digoxin and fondaparinux was well tolerated, with no significant changes in vital signs.

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Protein binding
94%In vitro Protein binding specificity against ATIII

[1]. Fondaparinux sodium: a selective inhibitor of factor Xa. Am J Health Syst Pharm. 2001 Nov 1;58 Suppl 2:S14-7.

[2]. Differential coagulation inhibitory effect of fondaparinux, enoxaparin and unfractionated heparin in cell models of thrombin generation. Blood Coagul Fibrinolysis. 2011 Jul;22(5):369-73.

[3]. Fondaparinux Sodium: Recent Advances in the Management of Thrombosis. J Cardiovasc Pharmacol Ther. 2023 Jan-Dec;28:10742484221145010.

Fondaparinux sodium is a synthetic pentasaccharide whose structure consists of five monosaccharide units, excluding the O-methyl group at the reducing end of the molecule. These monosaccharide units have the same sequence as the five monosaccharide units isolated by chemical or enzymatic cleavage of polysaccharide-glycosaminoglycan heparin and heparan sulfate. It is an anticoagulant. It is an aminosaccharide, oligosaccharide sulfate ester, and pentasaccharide derivative. Functionally, it is related to normethylfondaparinux sodium. It is the conjugate acid of fondaparinux sodium (10-). Fondaparinux sodium (Arixtra) is a synthetic anticoagulant composed of five monosaccharide units and an O-methyl group at the reducing end of the molecule. Its structure is similar to that of polysaccharide-glycosaminoglycan heparin and heparan sulfate (HS) when cleaved into monomer units. The monomer sequences in heparin and heparan sulfate (HS) are believed to form high-affinity binding sites for the natural anticoagulant factor antithrombin III (ATIII). Once ATIII binds to heparin or HS, its anticoagulant activity can be enhanced by 1000-fold. Fondaparinux sodium enhances the neutralizing effect of ATIII on activating factor X by 300-fold. Fondaparinux sodium can be used for: prevention of venous thromboembolism in patients undergoing lower extremity orthopedic surgery (e.g., hip fractures, hip replacements, and knee surgeries); prevention of venous thromboembolism in patients undergoing abdominal surgery at high risk of thromboembolic complications; treatment of deep vein thrombosis (DVT) and pulmonary embolism (PE); treatment of unstable angina (UA) and non-ST-segment elevation myocardial infarction (NSTEMI); and treatment of ST-segment elevation myocardial infarction (STEMI). Fondaparinux sodium is a factor Xa inhibitor. Its mechanism of action is as a factor Xa inhibitor. Fondaparinux sodium is a synthetic factor Xa inhibitor administered by injection as an anticoagulant for the treatment and prevention of venous thromboembolism. The incidence of elevated serum transaminases during fondaparinux sodium treatment is low, but it has not been found to be associated with clinically significant cases of specific liver injury.
Fondaparinux sodium is a synthetic glucosinolate with antithrombotic activity. Fondaparinux sodium selectively binds to antithrombin III, thereby enhancing the inherent neutralizing effect of antithrombin on activated factor X (factor Xa). Neutralizing factor Xa inhibits its activity and interrupts the blood coagulation cascade, thus preventing thrombin formation and thrombus formation.
Fondaparinux sodium is an oligosaccharide drug, with its clinical trial phase up to Phase IV (covering all indications). It was first approved in 2001 and currently has 6 approved indications and 12 investigational indications. This drug carries a black box warning from the U.S. Food and Drug Administration (FDA).
Synthetic pentasaccharides mediate the interaction between heparin and antithrombin and inhibit factor Xa; used for the prevention of postoperative venous thromboembolism.
Fondaparinux sodium is the sodium salt form of fondaparinux sodium, a synthetic glucosinolate with antithrombotic activity. Fondaparinux sodium selectively binds to antithrombin III, thereby enhancing the inherent neutralizing effect of antithrombin on activated factor X (factor Xa). Neutralization of factor Xa inhibits its activity and disrupts the blood coagulation cascade, thus preventing thrombin formation and thrombus formation. (NCI05)
A synthetic pentasaccharide that mediates the interaction between heparin and antithrombin and inhibits factor Xa; used for the prevention of postoperative venous thromboembolism.
See also: Fondaparinux sodium (containing the active ingredient).
Pharmaceutical Indications
1. 5 mg/0.3 ml and 2. 5 mg/0.5 ml injection: For the prevention of venous thromboembolic events (VTE) in adult patients undergoing major lower extremity orthopedic surgery (such as hip fracture, major knee surgery, or hip replacement). For the prevention of venous thromboembolism (VTE) in adult patients undergoing abdominal surgery who are considered to be at high risk of thromboembolic complications, such as patients undergoing abdominal tumor surgery. For the prevention of VTE in adult medical patients who are bedridden due to acute illness (e.g., heart failure and/or acute respiratory illness and/or acute infection or inflammatory disease) and are considered to be at high risk of VTE. For the treatment of adult patients with acute symptomatic spontaneous superficial vein thrombosis of the lower extremities without deep vein thrombosis. 2.5 mg/0.5 mL injection. For the treatment of adult patients with unstable angina or non-ST-segment elevation myocardial infarction (UA/NSTEMI) who are not suitable for emergency (<120 minutes) interventional treatment (PCI). For the treatment of adult patients with ST-segment elevation myocardial infarction (STEMI) who have received thrombolytic therapy or who initially did not receive other forms of reperfusion therapy. 5 mg/0.4 mL, 7.5 mg/0.6 mL, and 10 mg/0.8 mL injection. For the treatment of adult patients with acute deep vein thrombosis (DVT) and acute pulmonary embolism (PE), excluding hemodynamically unstable patients or patients requiring thrombolysis or pulmonary thrombectomy. 1.5 mg/0.3 mL and 2.5 mg/0.5 mL injections: for the prevention of venous thromboembolic events (VTE) in patients undergoing major lower extremity orthopedic surgery (such as hip fracture, major knee surgery, or hip replacement). For the prevention of VTE in patients undergoing abdominal surgery who are considered to be at high risk of thromboembolic complications, such as patients undergoing abdominal cancer surgery (see Section 5.1). For the prevention of VTE in medical patients who are bedridden due to acute illness (such as heart failure and/or acute respiratory illness and/or acute infection or inflammatory disease) and are considered to be at high risk of VTE. 2.5 mg/0.5 mL injection: for the treatment of patients with unstable angina or non-ST-segment elevation myocardial infarction (UA/NSTEMI) who are not suitable for emergency (<120 minutes) interventional treatment (PCI) (see Sections 4.4 and 5.1). This medication is indicated for the treatment of patients with ST-segment elevation myocardial infarction (STEMI) who have received thrombolytic therapy or who initially did not receive other forms of reperfusion therapy. 5 mg/0.4 ml, 7.5 mg/0.6 ml, and 10 mg/0.8 ml injections are indicated for the treatment of acute deep vein thrombosis (DVT) and acute pulmonary embolism (PE), except in hemodynamically unstable patients or those requiring thrombolysis or pulmonary embolization. Anticoagulants, including unfractionated heparin (UFH), enoxaparin, and fondaparinux, are approved drugs for acute coronary syndrome (ACS). Monocytes and monocyte-derived microparticles (MMPs) play an important procoagulant role in ACS by expressing high levels of tissue factor (TF), thereby triggering thrombin generation. This study aimed to compare the in vitro inhibitory effects of unfractionated heparin (UFH), enoxaparin, and fondaparinux in monocyte and matrix metalloproteinase (MMP) models. Human monocytes were activated with lipopolysaccharide (LPS) for 5 and 18 hours to obtain activated monocytes (ac-M) and mesenchymal monocytes (MMPs), respectively. Ac-M or MMPs were mixed with anemic platelet plasma containing different concentrations of anticoagulants to assess thrombin inhibition. Thrombin inhibition was dose-dependent, and the effects varied among different drugs: UFH showed the strongest inhibitory effect, while fondaparinux showed the weakest. The rate index was the most sensitive parameter. The IC50 values (anti-Xa factor IU/ml) of fondaparinux for ac-M and MMPs were 0.59±0.05 and 0.17±0.03, respectively. For enoxaparin, the rate index IC50 values for ac-M and MMPs were 0.27±0.03 and 0.19±0.02, respectively. Our data support that cell-induced thrombin generation assays may be a reliable alternative to determining patients' anticoagulation levels and can replace anti-Xa factor activity assays. [2] In summary, our study shows that three approved anticoagulants for ACS have different inhibitory effects, as assessed using a cell-induced thrombin generation model. Our results support the conclusions of the OASIS-5 sub-study that anticoagulation levels should be reduced. Our results clearly demonstrate the limitations of using anti-Xa factor activity assays to assess the effects of anticoagulant molecules. Thrombin generation assays are informative and sensitive methods, and standardization remains challenging despite ongoing efforts. Thrombin generation assays can be used to assess the pharmacodynamic effects of novel anticoagulants. Furthermore, thrombin generation assays can serve as a useful tool for assessing the levels required in Phase I and Phase II studies. [2]

C31H53N3O49S8

1508.26

1506.95133

C, 24.69; H, 3.54; N, 2.79; O, 51.98; S, 17.01

104993-28-4

5282448

Typically exists as solids at room temperature

0

19

52

30

91

3450

25

S(N[C@H]1[C@@H](O[C@@H]2[C@H](C(=O)O)O[C@H]([C@@H]([C@H]2O)OS(=O)(=O)O)O[C@@H]2[C@@H](COS(=O)(=O)O)O[C@@H]([C@@H]([C@H]2O)NS(=O)(=O)O)OC)O[C@H](COS(=O)(=O)O)[C@H]([C@@H]1OS(=O)(=O)O)O[C@H]1[C@@H]([C@H]([C@@H]([C@@H](C(=O)O)O1)O[C@@H]1[C@@H]([C@H]([C@@H]([C@@H](COS(=O)(=O)O)O1)O)O)NS(=O)(=O)O)O)O)(=O)(=O)O

Fondaparinux free acid; Fondaparinux; 104993-28-4; Natural heparin pentasaccharide; Arixtra; CHEBI:61033; UNII-J177FOW5JL; J177FOW5JL; HSDB 7845;

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)

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.)