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

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].

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].

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]

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.

Route of Elimination
After a 10-second bolus dose of 150 mg/m2 of ETHYOL, renal excretion of the parent drug and its two metabolites was low during the hour following drug administration, averaging 0.69%, 2.64% and 2.22% of the administered dose for the parent, thiol and disulfide, respectively.

Measurable concentrations of the active free thiol metabolite have been found in bone marrow cells 5 to 8 minutes after intravenous administration.

It is not known whether amifostine or its metabolites are distributed into breast milk.

Elimination /is/ primarily via rapid metabolism and uptake into tissues.

... Studies in rats using a single dose of amifostine showed that /with/ SC administration, there is no evidence of drug accumulation in either normal or tumor tissue, with tumor WR-1065 levels peaking just above the limits of quantitation during treatment. ... PMID:12577236

The pharmacokinetics of the cytoprotective agent amifostine (EthyolR; WR 2721) and its main metabolites (WR 1065 and the disulphides) were studied in patients participating in two phase I trials concerning carboplatin or cisplatin in combination with amifostine. Patients were treated with a single dose or three doses of amifostine (740 or 910 mg/sq m). The single or first dose was given as a 15 min iv infusion just before administration of the chemotherapeutic agent. The additional two infusions were administered 2 and 4 hr thereafter. Amifostine was rapidly cleared from the plasma, due to, at least in part, the fast conversion into WR 1065. A biphasic decrease with a final half-life of 0.8 hr was observed. The active metabolite WR 1065 was cleared from the plasma with a final half-life of 7.3 +/- 3.6 hr. The short initial half-life of WR 1065 can be explained by its fast uptake in tissues and the formation of disulphides. The disulphides were cleared with a final half-life of 8.4-13.4 hr and were detectable for at least 24 hr after treatment. They may serve as an exchangeable pool of WR 1065. The amifostine peak values at the end of each 15 min infusion did not accumulate in the multiple dosing schedule. For WR 1065 a trend towards an increase in the 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)], whereas a trend towards a small decrease was observed for the peak levels of the disulphides [C1,max: 184.2 +/- 12.6 uM, C2,max: 175.0 +/- 23.7 uM, C3,max: 166.0 +/- 17.2 uM, (n = 6)]. This latter finding might suggest a saturation of the disulphide formation or a change in the uptake or elimination of WR 1065, which would result in higher WR 1065 levels in plasma and tissues, after multiple doses of amifostine. PMID:9337685

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Metabolism / Metabolites
Amifostine is rapidly dephosphorylated by alkaline phosphatase in tissues primarily to the active free thiol metabolite and, subsequently, to a less active disulfide metabolite.

Amifostine is dephosphorylated by alkaline phosphatase in tissues primarily to the active free thiol metabolite and, subsequently, to a less active disulfide metabolite. Thomson/Micromedex. Drug Information for the Health Care Professional. Volume 1, Greenwood Village, CO. 2007., p. 90 Hazardous Substances Data Bank (HSDB) Within 1 hour after infusion of 740 to 910 mg per square meter of body surface area over 15 minutes or rapid intravenous injection of 150 mg per square meter body surface area over 10 seconds, urinary recovery of unchanged amifostine, of the disulfide metabolite, and the thiol metabolite accounts for only 0.69%, 2.22%, and 2.64%, respectively, of the dose.

This study investigated the metabolism of the radio- and chemoprotector compound, WR-2721 [amifostine; s-2-(3- aminopropylamino)ethylphosphorothioate], in the Balb/c mouse. ... It is known that /radiation/ protection requires conversion of the parent drug to its free thiol metabolite, WR-1065, in cultured cells. Because it is possible that metabolites of WR-1065 could be involved in protection and because thiols are metabolically very reactive molecules, we investigated the metabolism of WR-2721 using electrochemical detection-HPLC methods. The following are the major findings in this study: 1) WR-2721 drug was rapidly cleared from the bloodstream. Blood concentration of the parent drug decreased 10-fold 30 min after administration from the maximal observed value at 5 min 2) WR-1065 rapidly appeared in the perchloric acid (PCA)-soluble fraction of normal solid tissues. The highest WR-1065 concentrations in liver and kidney were 965 and 2195 mumol/kg, respectively, 10 min after parent drug administration, whereas for heart and small intestine the highest values were 739 and 410 mumol/kg at 30 min. 3) WR-1065 accumulated in the PCA-soluble fraction of two experimental tumors at a lower rate than for the other tissues. PMID:7895607

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Biological Half-Life
8 minutes
Approximately 8 minutes; less than 10% of amifostine remains in the plasma 6 minutes after drug administration.

The pharmacokinetics of the cytoprotective agent amifostine (EthyolR; WR 2721) and its main metabolites (WR 1065 and the disulphides) were studied in patients participating in two phase I trials concerning carboplatin or cisplatin in combination with amifostine. Patients were treated with a single dose or three doses of amifostine (740 or 910 mg/sq m). The single or first dose was given as a 15 min iv infusion just before administration of the chemotherapeutic agent. The additional two infusions were administered 2 and 4 hr thereafter. Amifostine was rapidly cleared from the plasma, due to, at least in part, the fast conversion into WR 1065. A biphasic decrease with a final half-life of 0.8 hr was observed. The active metabolite WR 1065 was cleared from the plasma with a final half-life of 7.3 +/- 3.6 hr. The short initial half-life of WR 1065 can be explained by its fast uptake in tissues and the formation of disulphides. The disulphides were cleared with a final half-life of 8.4-13.4 hr and were detectable for at least 24 hr after treatment. They may serve as an exchangeable pool of WR 1065. ...

Rat(ip): LD50: 418 mg/kg

Rat(im): LD50: 396 mg/kg

Mouse(po): LD50: 842 mg/kg

Mouse(ip): LD50: 321 mg/kg

Mouse(iv): LD50: 557 mg/kg

Mouse (im): LD50: 514 mg/kg

Dog(iv): LD Lo: 279 mg/kg

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Interactions
Repeated peroral administration of melatonin and ascorbic acid in a dose of 200 mg/kg 30 min before treatment with gammafos reduced its cumulative toxic effect. Succinic acid in a dose of 100 mg/kg was ineffective under these conditions. The cumulative death time for 50% animals receiving gammafos alone or in combination with melatonin, ascorbic acid, and succinic acid was 3.08, 4.29, 4.06, and 2.97 days, respectively. PMID:15455115

Amifostine may temporarily produce hypotension; antihypertensive or other potentially hypotension-producing medications should be discontinued 24 hours prior to amifostine administration; patients receiving antihypertensive therapy that cannot be discontinued should not receive amifostine.

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Antidote and Emergency Treatment
Immediate first aid: Ensure that adequate decontamination has been carried out. If patient is not breathing, start artificial respiration, preferably with a demand valve resuscitator, bag-valve-mask device, or pocket mask, as trained. Perform CPR if necessary. Immediately flush contaminated eyes with gently flowing water. Do not induce vomiting. If vomiting occurs, lean patient forward or place on the left side (head-down position, if possible) to maintain an open airway and prevent aspiration. Keep patient quiet and maintain normal body temperature. Obtain medical attention. /Poisons A and B/

Basic treatment: Establish a patent airway (oropharyngeal or nasopharyngeal airway, if needed). Suction if necessary. Watch for signs of respiratory insufficiency and assist ventilations if needed. Administer oxygen by nonrebreather mask at 10 to 15 L/min. Monitor for pulmonary edema and treat if necessary ... . Monitor for shock and treat if necessary ... . Anticipate seizures and treat if necessary ... . For eye contamination, flush eyes immediately with water. Irrigate each eye continuously with 0.9% saline (NS) during transport ... . Do not use emetics. For ingestion, rinse mouth and administer 5 mL/kg up to 200 mL of water for dilution if the patient can swallow, has a strong gag reflex, and does not drool ... . Cover skin burns with dry sterile dressings after decontamination ... . /Poisons A and B/

Advanced treatment: Consider orotracheal or nasotracheal intubation for airway control in the patient who is unconscious, has severe pulmonary edema, or is in severe respiratory distress. Positive-pressure ventilation techniques with a bag valve mask device may be beneficial. Consider drug therapy for pulmonary edema ... . Consider administering a beta agonist such as albuterol for severe bronchospasm ... . Monitor cardiac rhythm and treat arrhythmias as necessary ... . Start IV administration of D5W /SRP: "To keep open", minimal flow rate/. Use 0.9% saline (NS) or lactated Ringer's if signs of hypovolemia are present. For hypotension with signs of hypovolemia, administer fluid cautiously. Watch for signs of fluid overload ... . Treat seizures with diazepam or lorazepam ... . Use proparacaine hydrochloride to assist eye irrigation ... . /Poisons A and B/

[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 aminosulfhydryl compound. After dephosphorylation of amifostine by alkaline phosphatase to an active free sulfhydryl (thiol) metabolite, the thiol metabolite binds to and detoxifies cytotoxic platinum-containing metabolites of cisplatin and scavenges free radicals induced by cisplatin and ionizing radiation. The elevated activity of this agent in normal tissues results from both the relative abundance of alkaline phosphatase in normal tissues and the greater vascularity of normal tissues compared to tumor tissues.
A phosphorothioate proposed as a radiation-protective agent. It causes splenic vasodilation and may block autonomic ganglia.
See also: Amifostine (annotation moved to).

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.)