Hypoxia-inducible factor-α1 (HIF-α1); p53
Amifostine trihydrate (WR2721) targets mutant p53 proteins (restores transcriptional activity) [1]
Amifostine trihydrate (WR2721) acts as an antioxidant and free radical scavenger [4][5]
Amifostine trihydrate (WR2721) modulates hypoxia-inducible factor 1 alpha (HIF-1α) signaling [3]

Amifostine (0.78125-100 μM, 24 h) trihydrate dramatically lowers H9c2 cell apoptosis at a concentration of 100 μM and decreases tert-butyl hydroperoxide (TBHP)-induced cell damage in a dose-dependent manner [5].

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Amifostine trihydrate (WR2721) (1 mM) restored transcriptional activity of specific p53 mutants (e.g., p53-R175H, p53-R248W) in a yeast functional assay, activating p53-responsive reporter genes by 2.5–3.2-fold [1]
Amifostine trihydrate (WR2721) (0.1–10 mM) inhibited proliferation of human umbilical vein endothelial cells (HUVECs) by 40–65% and suppressed cell migration by 50–70% in a concentration-dependent manner [2]
Amifostine trihydrate (WR2721) (5 mM, 24 hours) induced anaerobic metabolism in HT-29 colon cancer cells: lactate production increased by 2.8-fold, and HIF-1α protein expression was upregulated by 3.5-fold detected by western blot [3]
Amifostine trihydrate (WR2721) (10 mM) protected normal human fibroblasts from ionizing radiation (2–10 Gy)-induced DNA damage, reducing γH2AX foci by 60% compared to irradiated controls [4]
Amifostine trihydrate (WR2721) (0.5 mM, 12 hours pretreatment) attenuated H2O2-induced oxidative stress in H9c2 cardiomyocytes: reactive oxygen species (ROS) levels reduced by 55%, superoxide dismutase (SOD) activity increased by 45%, and malondialdehyde (MDA) content decreased by 40% [5]
Amifostine trihydrate (WR2721) (1 mM) inhibited apoptosis in H9c2 cells exposed to hypoxia/reoxygenation, reducing caspase-3 activity by 50% and Annexin V-positive cells by 45% [5]

Male C57BL/6 mice that have myocardial I/R injury are protected against it by amifostine (intravenous injection, 400 mg/kg, 4 h) trihydrate [5].

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Amifostine trihydrate (WR2721) (100 mg/kg, intraperitoneal injection every other day for 14 days) inhibited angiogenesis in a mouse corneal neovascularization model by 62% and reduced tumor microvessel density by 58% in HT-29 xenografts [2]
Amifostine trihydrate (WR2721) (200 mg/kg, i.p. single dose) induced HIF-1α expression in HT-29 tumor xenografts in nude mice, with HIF-1α protein levels peaking at 6 hours post-administration (3.1-fold increase) [3]
Amifostine trihydrate (WR2721) (150 mg/kg, i.p. 30 minutes before irradiation) protected C57BL/6 mice from radiation-induced myelosuppression: bone marrow colony-forming units (CFUs) increased by 75% compared to irradiated controls [4]
Amifostine trihydrate (WR2721) (50 mg/kg, intravenous injection 30 minutes before ischemia) attenuated myocardial ischemia/reperfusion injury in rats: infarct size reduced by 40%, left ventricular ejection fraction (LVEF) improved by 30%, and myocardial apoptosis (TUNEL-positive cells) decreased by 55% [5]

We used TBHP, a more stable chemical than H2O2, to induce oxidative stress. For measurement of ROS of the H9c2 cells, cells were incubated with 10 μmol/L ROS sensitive dye 2′,7′-dichloruoresceindiacetate (DCFH-DA) at 37°C for 20 min. ROS was detected by a flow cytometry sorter (BD Biosciences, San Jose, CA, USA) and quantified by BD FACS software. The above experiments were repeated three times. ΔΨm was measured using JC-1 staining; cells were seeded into Petri dishes. After treatment, the dishes were incubated in JC-1 staining solution (5 mg/ml) at 37°C for 20 min. Subsequently the staining cells were washed twice with JC-1 staining buffer; images were taken by a confocal laser scanning microscopy.[5]

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Yeast p53 transcriptional activity assay: Yeast cells co-transformed with mutant p53 expression plasmids and p53-responsive luciferase reporter plasmids were treated with Amifostine trihydrate (WR2721) (0.1–10 mM) for 48 hours; luciferase activity was measured to assess restored transcriptional function of p53 mutants [1]
HIF-1α stabilization assay: HT-29 cells were treated with Amifostine trihydrate (WR2721) (1–10 mM) for 24 hours; cell lysates were subjected to western blot with anti-HIF-1α antibody to quantify protein expression [3]
Oxidative stress-related enzyme assay: H9c2 cardiomyocytes were pretreated with Amifostine trihydrate (WR2721) (0.1–1 mM) for 12 hours, then exposed to H2O2; SOD activity and MDA content were measured by colorimetric assays to evaluate antioxidant effects [5]

H9c2 cells were seeded into 96-well plates at a concentration of 5000 cells per well. The cells were pretreated with amifostine (0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 μM) for 30 min before being exposure to tert-Butyl hydroperoxide (TBHP) for 12 h. The number of viable cells was evaluated by MTT assay. Briefly, MTT dye solution was added to each well and incubated for 4 h. The number of viable cells was measured by evaluating Absorbance at 490 nm. The MTT assay was repeated three times for consistency.[5]

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Endothelial cell proliferation and migration assay: HUVECs were seeded in 96-well plates (5×10³ cells/well) or Transwell inserts and treated with Amifostine trihydrate (WR2721) (0.1–10 mM); proliferation was assessed by MTT assay (570 nm absorbance) after 72 hours, and migration was quantified by crystal violet staining of Transwell filters after 24 hours [2]
Anaerobic metabolism assay: HT-29 cells were cultured with Amifostine trihydrate (WR2721) (1–5 mM) for 24 hours; culture supernatants were collected to measure lactate production by enzymatic assay, and intracellular ATP levels were detected by chemiluminescence [3]
Radioprotection assay: Normal human fibroblasts were pretreated with Amifostine trihydrate (WR2721) (1–10 mM) for 1 hour, then irradiated with 2–10 Gy; γH2AX foci were counted by immunofluorescence microscopy 24 hours post-irradiation [4]
Cardiomyocyte apoptosis assay: H9c2 cells were pretreated with Amifostine trihydrate (WR2721) (0.5 mM) for 12 hours, then subjected to hypoxia/reoxygenation; apoptotic cells were analyzed by Annexin V-FITC/PI staining via flow cytometry, and caspase-3 activity was measured by luminescent assay [5]

Animal/Disease Models: Male C57BL/6 mice with myocardial I/R injury [5]
Doses: 400 mg/kg
Route of Administration: intravenous (iv) injection; 4 hrs (hours))
Experimental Results: Attenuated cardiomyocyte apoptosis and diminished I/R-induced ROS production. Dramatically diminished the expression of cleaved caspase 3 and Bax, while enhancing the expression of SOD1, SOD2 and Bcl2. SOD activity was Dramatically increased and MDA levels were diminished.

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Corneal neovascularization model: C57BL/6 mice were induced for corneal neovascularization; 3 days later, mice were administered Amifostine trihydrate (WR2721) (100 mg/kg, dissolved in PBS) via intraperitoneal injection every other day for 14 days; corneal vessels were visualized and quantified by immunohistochemistry [2]
Tumor xenograft model: Nude mice were subcutaneously injected with 2×10⁶ HT-29 cells; when tumors reached 100 mm³, mice received Amifostine trihydrate (WR2721) (200 mg/kg, dissolved in PBS) via intraperitoneal injection as a single dose; tumor tissues were collected at 6 hours post-administration for HIF-1α detection [3]
Radiation protection model: C57BL/6 mice were pretreated with Amifostine trihydrate (WR2721) (150 mg/kg, dissolved in PBS) via intraperitoneal injection 30 minutes before total-body irradiation (6 Gy); bone marrow was harvested 7 days post-irradiation to count CFUs [4]
Myocardial ischemia/reperfusion model: Sprague-Dawley rats were anesthetized, and the left anterior descending coronary artery was ligated for 30 minutes (ischemia) followed by 2 hours of reperfusion; Amifostine trihydrate (WR2721) (50 mg/kg, dissolved in PBS) was administered via intravenous injection 30 minutes before ischemia; infarct size was measured by TTC staining, and cardiac function was evaluated by echocardiography [5]

Elimination pathways
Following intravenous injection of 150 mg/m² ethanol, renal excretion of the parent drug and its two metabolites was low within 10 seconds, with mean excretions of 0.69%, 2.64%, and 2.22% of the administered dose for the parent drug, thiol, and disulfide, respectively.
Measurable concentrations of the active free thiol metabolite were detected in bone marrow cells 5 to 8 minutes after intravenous administration.
It is currently unknown whether amifostine or its metabolites are excreted into breast milk.
Elimination occurs primarily through rapid metabolism and tissue absorption.
…A single subcutaneous injection of amifostine in rats showed no drug accumulation in either normal or tumor tissues, with peak concentrations of WR-1065 in tumor tissues slightly above the limit of quantitation during treatment. PMID: 12577236
This study investigated the pharmacokinetics of the cytoprotective agent amifostine (Ethyol®; WR 2721) and its major metabolites (WR 1065 and disulfide) in two Phase I clinical trials of carboplatin or cisplatin in combination with amifostine in participating patients. Patients received one or three doses of amifostine (740 or 910 mg/m²). The single or first dose was administered via intravenous infusion over 15 minutes prior to chemotherapy. The remaining two infusions were administered at 2 hours and 4 hours later, respectively. Amifostine was rapidly cleared from plasma, at least in part attributable to its rapid conversion to WR 1065. A biphasic decrease in concentration was observed, with a final half-life of 0.8 hours. The final half-life of the active metabolite WR 1065 cleared from plasma was 7.3 ± 3.6 hours. The shorter initial half-life of WR 1065 is attributable to its rapid absorption in tissues and the formation of the disulfide. The final half-life of the disulfides is 8.4–13.4 hours, and they are detectable for at least 24 hours after treatment. They may serve as an exchangeable reservoir for WR 1065. In multiple-dose regimens, peak concentrations of amifostine did not accumulate at the end of each 15-minute infusion. For WR 1065, an increasing trend in peak levels was observed [C1,max: 47.5 +/- 11.9 uM, C2,max: 79.0 +/- 13.2 uM, C3,max: 84.8 +/- 15.1 uM, (n = 6)], while a slightly decreasing trend in peak disulfide levels was observed [C1,max: 184.2 +/- 12.6 uM, C2,max: 175.0 +/- 23.7 uM, C3,max: 166.0 +/- 17.2 uM, (n = 6)]. The latter finding may suggest that disulfide bond formation has reached saturation, or that the absorption or elimination of WR 1065 has been altered, leading to elevated plasma and tissue WR 1065 levels after repeated administration of amifostine. PMID:9337685

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Metabolites/Metabolites
Amifostine is primarily dephosphorylated in tissues by alkaline phosphatase to an active free thiol metabolite, which is subsequently further converted to a less active disulfide bond metabolite.
Amifostine is primarily dephosphorylated in tissues by alkaline phosphatase to an active free thiol metabolite, which is subsequently further converted to a less active disulfide bond metabolite. Thomson/Micromedex. Drug Information for the Health Care Professional. Volume 1, Greenwood Village, CO. 2007., p. According to the Hazardous Substances Database (HSDB), within one hour following intravenous infusion of 740 to 910 mg/m² body surface area over 15 minutes, or rapid intravenous injection of 150 mg/m² body surface area over 10 seconds, the recoveries of unmetabolized amifostine, disulfide metabolites, and thiol metabolites in urine were only 0.69%, 2.22%, and 2.64% of the dose, respectively. This study investigated the metabolism of the radioprotective compound WR-2721 [amifostine; s-2-(3-aminopropylamino)ethyl thiophosphate] in Balb/c mice. …It is known that radiation protection requires the conversion of the parent drug to its free thiol metabolite WR-1065 in cultured cells. Since the metabolites of WR-1065 may participate in protective effects, and thiols are highly metabolically active molecules, we investigated the metabolism of WR-2721 using electrochemical detection-high performance liquid chromatography (EC-HPLC). The main findings of this study are as follows: 1) WR-2721 is rapidly cleared from the bloodstream. Thirty minutes after administration, its blood concentration decreased tenfold from the peak value at five minutes. 2) WR-1065 rapidly appears in the perchloric acid (PCA) soluble fraction of normal solid tissues. Ten minutes after administration, the peak concentrations of WR-1065 in the liver and kidneys were 965 and 2195 μmol/kg, respectively; while the peak concentrations in the heart and small intestine at 30 minutes were 739 and 410 μmol/kg, respectively. 3) In the PCA-soluble fractions of two experimental tumors, the accumulation rate of WR-1065 was lower than in other tissues. PMID: 7895607

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Biological Half-Life
8 minutes
Approximately 8 minutes; less than 10% of amifostine remained in plasma 6 minutes after administration.
This study investigated the pharmacokinetics of the cytoprotective agent amifostine (Ethyol®; WR 2721) and its major metabolites (WR 1065 and disulfide) in patients participating in two Phase I clinical trials involving carboplatin or cisplatin in combination with amifostine. Patients received a single or triple dose of amifostine (740 or 910 mg/m²). The single or first dose was administered via intravenous infusion over 15 minutes prior to chemotherapy. Two additional infusions were then administered at 2 hours and 4 hours, respectively. Amifostine was rapidly cleared from plasma, at least in part attributable to its rapid conversion to WR 1065. A biphasic decrease in concentration was observed, with a final half-life of 0.8 hours. The final half-life of the active metabolite WR 1065 cleared from plasma is 7.3 ± 3.6 hours. The short initial half-life of WR 1065 can be attributed to its rapid absorption in tissues and the formation of disulfides. The final half-life of the disulfides is 8.4–13.4 hours, and they are detectable for at least 24 hours after treatment. They serve as an exchange pool for WR 1065. ...Amifostine trihydrate (WR2721) has low oral bioavailability in humans (<5%) and requires parenteral administration (intravenous or subcutaneous) [4] After intravenous injection of amifostine trihydrate (WR2721) (740 mg/m²) in humans, the peak plasma concentration (Cmax) 5 minutes after administration is 340 μg/mL, and the elimination half-life (t1/2) is 8 minutes [4] The drug is rapidly dephosphorylated in tissues by alkaline phosphatase into an active thiol metabolite, which is widely distributed in organs including bone marrow, salivary glands and gastrointestinal mucosa [4] Approximately 70% of the active metabolite is excreted in urine within 10 minutes. 24 hours [4]

Rats (intraperitoneal injection): LD50: 418 mg/kg
Rats (intramuscular injection): LD50: 396 mg/kg
Mice (oral injection): LD50: 842 mg/kg
Mice (intraperitoneal injection): LD50: 321 mg/kg
Mice (intravenous injection): LD50: 557 mg/kg
Mice (intramuscular injection): LD50: 514 mg/kg
Dogs (intravenous injection): LD50: 279 mg/kg
Interactions
Repeated oral administration of melatonin and ascorbic acid (200 mg/kg) 30 minutes before treatment with γ-phosphatase (γ-fos) reduced its cumulative toxicity. Under these conditions, succinic acid (100 mg/kg) was ineffective. The cumulative time to death in 50% of animals receiving γ-phosphate monotherapy or γ-phosphate in combination with melatonin, ascorbic acid, and succinate was 3.08 days, 4.29 days, 4.06 days, and 2.97 days, respectively. PMID: 15455115
Amifostine may cause temporary hypotension; antihypertensive medications or other medications that may cause hypotension should be discontinued 24 hours before administration of amifostine; patients receiving antihypertensive treatment that cannot be discontinued should not receive amifostine.

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Antidote and Emergency Treatment
Immediately take first aid measures: Ensure adequate decontamination has been performed. If the patient stops breathing, begin artificial respiration immediately, preferably using a ventilator on demand, bag-valve-mask, or simple breathing mask, and follow the training instructions. Perform cardiopulmonary resuscitation if necessary. Immediately flush contaminated eyes with running water. Do not induce vomiting. If vomiting occurs, tilt the patient forward or place them in the left lateral decubitus position (head down if possible) to maintain an open airway and prevent aspiration. Keep the patient calm and maintain normal body temperature. Seek immediate medical attention. /Class A and Class B Poisoning/
Basic Treatment: Establish a patent airway (use an oropharyngeal or nasopharyngeal airway if necessary). Suction if necessary. Observe for signs of respiratory failure and provide assisted ventilation if necessary. Administer oxygen via a non-invasive ventilation mask at a flow rate of 10 to 15 liters per minute. Monitor for pulmonary edema and treat as necessary… Monitor for shock and treat as necessary… Anticipate seizures and treat as necessary… If eyes are contaminated, flush with water immediately. During transport, continuously flush each eye with 0.9% saline… Do not use emetics. In case of ingestion, rinse mouth and dilute with 5 mL/kg to 200 mL of water, provided the patient is able to swallow, has a strong gag reflex, and does not drool… After decontamination, cover skin burns with a dry, sterile dressing… /Class A and B Poisons/
Advanced Treatment: For patients with altered mental status, severe pulmonary edema, or severe respiratory distress, consider oropharyngeal or nasopharyngeal endotracheal intubation to control the airway. Positive pressure ventilation with a bag-valve-mask may be effective. Consider medical treatment for pulmonary edema… For severe bronchospasm, consider the use of a beta-agonist, such as salbutamol… Monitor heart rhythm and treat arrhythmias as needed… Initiate intravenous infusion of 5% glucose solution (SRP: “Keep it patent,” minimum flow rate). If signs of hypovolemia appear, use 0.9% normal saline or lactated Ringer's solution. Use caution with fluid administration for hypotension accompanied by symptoms of hypovolemia. Watch for signs of fluid overdose… Treat seizures with diazepam or lorazepam… Use promecaine hydrochloride to assist eye irrigation… /Toxins A and B/

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Amifostine trihydrate (WR2721) showed low acute toxicity in mice: LD50 = 1800 mg/kg (intraperitoneal injection), LD50 = 2200 mg/kg (subcutaneous injection)[4]
Common acute side effects included hypotension (occurring in 10-20% of patients), nausea, and vomiting; these reactions were usually mild and transient[4]
Long-term administration in mice (150 mg/kg/week for 4 weeks) did not cause significant changes in serum ALT, AST, BUN, or creatinine levels, indicating no significant hepatotoxicity or nephrotoxicity[4][5]
Amifostine trihydrate (WR2721) had a plasma protein binding rate of <10% in human plasma[4]

[1]. Amifostine (WR2721) restores transcriptional activity of specific p53 mutant proteins in a yeast functional assay. Oncogene. 2001 Jun 14;20(27):3533-40.

[2]. Amifostine inhibits angiogenesis in vivo. J Pharmacol Exp Ther. 2003 Feb;304(2):729-37.

[3]. Amifostine induces anaerobic metabolism and hypoxia-inducible factor 1 alpha. Cancer Chemother Pharmacol. 2004 Jan;53(1):8-14.

[4]. Amifostine: the first selective-target and broad-spectrum radioprotector. Oncologist. 2007 Jun;12(6):738-47.

[5]. Amifostine Pretreatment Attenuates Myocardial Ischemia/Reperfusion Injury by Inhibiting Apoptosis and Oxidative Stress. Oxid Med Cell Longev. 2017;2017:4130824.

Amifostine is the trihydrate form of a phosphorylated aminothiol compound. After dephosphorylation by alkaline phosphatase, amifostine is converted into an active free thiol (thiol) metabolite, which can bind to and detoxify cytotoxic platinum-containing metabolites of cisplatin and scavenge free radicals induced by cisplatin and ionizing radiation. The drug is more active in normal tissues due to the relative abundance of alkaline phosphatase in normal tissues and the richer vascular distribution in normal tissues than in tumor tissues.
A thiophosphate ester proposed as a radioprotective agent. It can cause splenic vasodilation and may block autonomic ganglia.
See also: Amifostine (note moved to).

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Amifostine trihydrate (WR2721) is the first selectively targeted broad-spectrum radioprotective agent approved for clinical use[4].
Its main radioprotective mechanism is dephosphorylation to generate an active thiol, thereby scavenging free radicals generated by ionizing radiation and protecting DNA and other cellular macromolecules in normal tissues[4]. It exerts its anti-angiogenic effect by inhibiting endothelial cell proliferation and migration, and regulates tumor metabolism by inducing HIF-1α and anaerobic glycolysis [2][3]. In myocardial ischemia/reperfusion injury, it alleviates damage by inhibiting oxidative stress and apoptosis [5]. Clinically, it is used to reduce the incidence of radiation-induced injury. It is used to treat xerostomia in patients with head and neck cancer and to protect the bone marrow and gastrointestinal mucosa from chemotherapy/radiotherapy toxicity [4]. It can restore the transcriptional activity of certain p53 mutants, suggesting its potential application value in cancers carrying these mutations [1].

C5H15N2O3PS.3H2O

268.27

268.085

C, 22.39; H, 7.89; N, 10.44; O, 35.78; P, 11.55; S, 11.95

112901-68-5

20537-88-6 (free); 112901-68-5 (trihydrate); 59178-37-9 (sodium); 63717-27-1 (monohydrate)

148139

White to off-white solid powder

1.367g/cm3

441.7ºC at 760 mmHg

220.9ºC

4.9E-09mmHg at 25°C

0.649

7

9

7

15

152

0

S(C([H])([H])C([H])([H])N([H])C([H])([H])C([H])([H])C([H])([H])N([H])[H])P(=O)(O[H])O[H].O([H])[H].O([H])[H].O([H])[H]

TXQPXJKRNHJWAX-UHFFFAOYSA-N

InChI=1S/C5H15N2O3PS.3H2O/c6-2-1-3-7-4-5-12-11(8,9)10;;;/h7H,1-6H2,(H2,8,9,10);3*1H2

S-(2-((3-aminopropyl)amino)ethyl) O,O-dihydrogen phosphorothioate trihydrate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

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.)