Hypoxia-inducible factor-α1 (HIF-α1); p53

Amifostine (0.78125-100 μM, 24 h) dramatically lowers H9c2 cell apoptosis at a concentration of 100 μM and decreases tertiary tetrahydroperoxide (TBHP)-induced cell damage in a dose-wise manner [5].

Amifostine (intravenous injection, 400 mg/kg, 4 hours) exerts a protective effect on myocardial I/R damage in male C57BL/6 mice [5].

Researchers 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]

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]

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.

Elimination pathways:
Following intravenous injection of 150 mg/m² ethanol, renal excretion of the parent drug and its two metabolites is low within 10 seconds. Within one hour post-administration, the average renal excretion of the parent drug, thiols, and disulfides is 0.69%, 2.64%, and 2.22% of the administered dose, 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 primarily occurs through rapid metabolism and tissue absorption.

A single-dose subcutaneous amifostine study in rats showed no drug accumulation in either normal or tumor tissues, with peak concentrations of WR-1065 in tumors slightly above the limit of quantitation. 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 patients participating in two Phase I clinical trials of 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. The remaining two infusions were administered at 2 hours and 4 hours, respectively. Amifostine was rapidly cleared from plasma, at least in part due to its rapid conversion to WR 1065. A biphasic decline 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 can be attributed 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 WR 1065 levels in plasma and tissues after repeated administration of amifostine. PMID: 9337685

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Metabolism/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. 90. Hazardous Substances Database (HSDB). Following a rapid intravenous infusion of 740 to 910 mg/m² body surface area over 15 minutes, or a 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 within one hour 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 after administration; 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 may serve as an exchange pool for WR 1065.

Rats (intraperitoneal injection): LD50: 418 mg/kg
Rats (intramuscular injection): LD50: 396 mg/kg
Mice (oral): 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

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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 treated with gamma-phosphate monotherapy or in combination with melatonin, ascorbic acid, and succinate were 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 demand ventilator, 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… Consider the use of a beta-agonist (such as salbutamol) for severe bronchospasm… Monitor cardiac rhythm and treat arrhythmias if necessary… Initiate intravenous infusion of 5% glucose solution (SRP: “Keep it patent,” minimum flow rate). If signs of hypovolemia appear, use 0.9% normal saline (NS) or lactated Ringer's solution. Use fluids with caution in cases of hypotension with signs of hypovolemia. Watch for signs of fluid overdose… Use diazepam or lorazepam to treat seizures… Use promecaine hydrochloride to assist eye irrigation… /Class A and B Poisons/

[1]. D Maurici, et al. Amifostine (WR2721) restores transcriptional activity of specific p53 mutant proteins in a yeast functional assay. Oncogene. 2001 Jun 14;20(27):3533-40.
[2]. Efstathia Giannopoulou, et al. Amifostine inhibits angiogenesis in vivo. J Pharmacol Exp Ther. 2003 Feb;304(2):729-37.
[3]. Michael I Koukourakis, et al. Amifostine induces anaerobic metabolism and hypoxia-inducible factor 1 alpha. Cancer Chemother Pharmacol. 2004 Jan;53(1):8-14.
[4]. John R Kouvaris, et al. Amifostine: the first selective-target and broad-spectrum radioprotector. Oncologist. 2007 Jun;12(6):738-47.
[5]. Shao-Ze Wu, et al. Amifostine Pretreatment Attenuates Myocardial Ischemia/Reperfusion Injury by Inhibiting Apoptosis and Oxidative Stress. Oxid Med Cell Longev. 2017;2017:4130824.

phosphorothioate proposed for use as a radiation protectant. It can cause splenic vasodilation and may block autonomic ganglia.
See also: Amifostine (note moved to).

C5H15N2O3PS.H2O

232.24

232.065

C, 25.86; H, 7.38; N, 12.06; O, 27.56; P, 13.34; S, 13.80

63717-27-1

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

83996

Typically exists as solid at room temperature

441.7ºC at 760 mmHg

220.9ºC

0.777

5

7

7

13

152

0

O.NCCCNCCSP(=O)(O)O

CWHOHHKTRJUFTR-UHFFFAOYSA-N

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

2-(3-aminopropylamino)ethylsulfanylphosphonic acid;hydrate

Amifostine hydrate; 63717-27-1; Amifostine monohydrate; Amifostinum; Amifostina; 2-(3-aminopropylamino)ethylsulfanylphosphonic acid hydrate; 63717-27-1 (monohydrate); L693H6MM64;

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.)