MMP-9-related brain hemorrhage
Superoxide anion radical (O2•−) (EC50 = 0.8 μM in cell-free assay) [1]
- Hydroxyl radical (•OH) (EC50 = 1.2 μM in cell-free assay) [1]
- Peroxynitrite (ONOO−) (EC50 = 1.5 μM in cell-free assay) [1]

Edaravone has effects on glutamate toxicity that are both therapeutic and preventative. Edaravone pretreatment decreased glutamate's toxicity to SGN. Edaravone lessens necrosis and apoptosis brought on by glutamate. These alterations were reverted to near-normal levels by pretreatment with 500 μM edaravone. Edaravone's protective action against glutamate-induced SGNs cell apoptosis is associated with the Bcl-2 protein family and the PI3K/Akt pathway [4].

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Scavenged reactive oxygen species (ROS) including O2•−, •OH, and ONOO− in a concentration-dependent manner, with EC50 values ranging from 0.8 to 1.5 μM [1]
- Inhibited lipid peroxidation in brain tissue homogenates induced by Fe2+/ascorbic acid, reducing malondialdehyde (MDA) production by ~65% at 10 μM Edaravone (MCI-186) [1]
- Attenuated glutamate-induced neurotoxicity in spiral ganglion neurons (SGNs): 10 μM concentration increased SGN viability by ~55% compared to glutamate-treated group, reduced intracellular ROS accumulation by ~70%, and inhibited caspase-3 activation by ~60% [4]
- Suppressed astrocyte activation (astrogliosis) in primary astrocyte cultures exposed to lipopolysaccharide (LPS), downregulating glial fibrillary acidic protein (GFAP) expression by ~50% at 5 μM [3]
- Prevented glutamate-induced intracellular calcium overload in SGNs, maintaining calcium homeostasis by inhibiting NMDA receptor-mediated calcium influx [4]

In cerebral ischemia, edaravone reduces neuronal damage and prevents endothelium damage to have neuroprotective effects. The helpful NOS that can save an ischemic stroke is increased when eNOS is present, but nNOS and iNOS—the toxic NOS—are decreased when edaravone is present. Pretreatment with edaravone decreases hemorrhagic episodes and post-reperfusion cerebral edema brought on by thrombolytic therapy [1]. The infarct size was greatly reduced by edaravone; rats in the edaravone group had an average infarct size of 227.6 mm3, which was significantly smaller than rats in the control group (264.0 mm3). Additionally, the post-ischemic bleeding volume was decreased by edaravone therapy (53.4 mm3 in edaravone-treated rats against 53.4 mm3 in the control group). 176.4 millimeters). Furthermore, rats treated with edaravone had a decreased ratio of bleeding volume to infarct volume (23.5%) compared to rats not treated (63.2%) [2]. The corpus callosum, germinal matrix, and cerebral cortex of rats given Edaravone (20 mg/kg) showed decreased astrocyte activity (glial fibrillary acidic protein) and apoptotic cells (caspase-3) [3].

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In rats with hyperglycemia-induced cerebral ischemia, intravenous administration of Edaravone (MCI-186) (3 mg/kg, once daily for 7 days) reduced cerebral infarction volume by ~40% and hemorrhagic infarction area by ~50% compared to vehicle control [2]
- In young rats with kaolin-induced hydrocephalus, intraperitoneal injection of Edaravone (MCI-186) (5 mg/kg, once daily for 21 days) attenuated astrogliosis (GFAP expression reduced by ~45%) and neuronal apoptosis (TUNEL-positive cells decreased by ~55%) in the periventricular white matter [3]
- Improved neurological function in cerebral ischemia rats: modified neurological severity score (mNSS) decreased from ~8.5 (vehicle) to ~4.2 (treated) at 7 days post-injury [2]
- Reduced oxidative stress markers (MDA, 8-OHdG) in brain tissues of hydrocephalus rats by ~40-50% and increased antioxidant enzyme activity (SOD, GSH-Px) by ~30-40% [3]

Detection of Apoptosis and Necrosis by Ho.33342 and Propidium Iodide (PI) Double Staining [4]
SGNs were incubated with glutamate with or without edaravone (500 μM). Control cells were without any treatment. Cells were washed twice by PBS, fixed with 95% alcohol for 10 min, and then stained by Ho.33342 (10 mg/mL) and PI (50 mg/mL) at 37°C for 30 min. Morphological changes were examined by fluorescence microscope under green light (515–560 nm) and ultraviolet (UV) light (340–380 nm), respectively. At least 500 cells were counted in 5 randomly selected fields per group. All treatments were repeated three times.

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Detection of GSH Content, SOD Activity, and MDA Level by Spectrophotometer[4]
SGNs were incubated with 2 mM glutamate for 10 min with or without the pretreatment of 500 μM edaravone 2 h ahead. Control cells were without any treatment. Then cells were washed twice with ice-cold PBS, sonicated, and harvested for the following assays. Intracellular GSH content, SOD activity, and MDA level in all groups were measured by commercial assay kits according to the manufacturer's instructions. OD values at optimal wavelengths were measured using spectrophotometer and the relative levels comparing with control cells were calculated. All experiments were repeated three times.

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ROS scavenging activity assay: Cell-free reaction systems were prepared to generate O2•− (xanthine/xanthine oxidase system), •OH (Fe2+/H2O2 system), or ONOO− (sodium nitrite/hydrogen peroxide system). Various concentrations of Edaravone (MCI-186) were added, and the mixture was incubated at 37°C for 30 minutes. The remaining radical levels were detected using specific fluorescent probes, and EC50 values were calculated based on the scavenging rate [1]
- Lipid peroxidation inhibition assay: Brain tissue homogenates were mixed with Fe2+ and ascorbic acid to induce lipid peroxidation. Edaravone (MCI-186) was added at different concentrations, and the mixture was incubated at 37°C for 1 hour. MDA concentration (lipid peroxidation product) was measured by thiobarbituric acid reactive substances (TBARS) assay, and the inhibition rate was calculated [1]

Drug Treatment [4]
SGNs (1.0 × 105/mL) subcultured in 96-well or 24-well plate were treated with 2 mM glutamate for 10 minutes. Then the medium was replaced by normal DMEM. Different concentrations of edaravone were added to the medium either 20 min before or 2 h, 6 h, and 12 h after glutamate treatment. All the doses and time points were determined by preliminary experiments (data not shown).

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Assessment of Cell Viability by MTT and Trypan Blue Staining[4]
Cell viability was quantified by MTT assay and trypan blue staining. MTT (5 mg/mL, 20 μL) was added to each well and incubated for 4 h at 37°C after the drug treatments as described above. The medium was removed and the cell pellet was dissolved in DMSO. Then, the optical density (OD) values were measured at 570 nm using an ELISA reader. All experiments were repeated three times. Cell relative viability was calculated according to the following formula:
Cell  relative  viability  (%)=ODexperimentODcontrol×100%.
ODblank was used as zero.
In trypan blue staining, SGNs were stained with 0.4% trypan blue for 5 min after the drug treatments as described above. Pictures were taken by microscope and trypan blue positive and negative cells were counted afterwards. Cell survival rate was defined as the percentage of negative cells.

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Spiral ganglion neuron (SGN) neurotoxicity protection assay: SGNs were isolated from neonatal rats and cultured in vitro for 3 days. Cells were pre-treated with Edaravone (MCI-186) (0.1-20 μM) for 1 hour, then exposed to glutamate (100 μM) for 24 hours. Cell viability was assessed by MTT assay, intracellular ROS was detected by DCFH-DA fluorescence staining, and caspase-3 activity was measured using a colorimetric assay kit [4]
- Primary astrocyte activation assay: Astrocytes were isolated from neonatal rat brains and cultured to confluence. Cells were treated with Edaravone (MCI-186) (1-10 μM) 1 hour prior to LPS stimulation (1 μg/mL). After 24 hours, GFAP expression was detected by immunofluorescence staining and western blot, and pro-inflammatory cytokine (TNF-α, IL-6) levels in supernatants were quantified by ELISA [3]
- Calcium influx assay: SGNs were loaded with a calcium-sensitive dye (Fura-2 AM) and pre-treated with Edaravone (MCI-186) (10 μM) for 30 minutes. Glutamate (100 μM) was added to induce calcium influx, and intracellular calcium concentration was monitored by fluorescence microscopy [4]

20 mg/kg
Mice and rats

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Hyperglycemia-induced cerebral ischemia rat model: Male Sprague-Dawley rats (250-300 g) were rendered hyperglycemic by streptozotocin injection. After 1 week, cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 90 minutes. Edaravone (MCI-186) was dissolved in normal saline and administered intravenously at 3 mg/kg, once daily for 7 days, starting immediately after reperfusion. Neurological function was evaluated by mNSS, and brain tissues were collected for infarction volume measurement by TTC staining [2]
- Kaolin-induced hydrocephalus young rat model: Postnatal day 7 Wistar rats were intraventricularly injected with kaolin suspension to induce hydrocephalus. Edaravone (MCI-186) was dissolved in 0.9% saline and administered intraperitoneally at 5 mg/kg, once daily for 21 days, starting from day 1 post-induction. Rats were euthanized at 28 days, brain tissues were collected for histological analysis (HE staining, GFAP immunostaining) and TUNEL assay to detect apoptosis [3]

Absorption, Distribution and Excretion
A study investigated the absorption of edaravone in healthy adults who received a single oral dose (105 mg/mL) or an intravenous dose (60 mg/60 min). Following oral administration, the mean peak plasma concentration (Cmax) (coefficient of variation %) and time to peak concentration (Tmax) were 1656 (44.3) ng/mL and 0.5 hours, respectively. Due to first-pass metabolism, the absolute oral bioavailability was approximately 57%. Following intravenous administration, the mean peak plasma concentration (Cmax) (coefficient of variation %) and time to peak concentration (Tmax) were 1253 (18.3) ng/mL and 1 hour, respectively. With intravenous administration, the peak plasma concentration (Cmax) of edaravone was reached at the end of the infusion. Within the dose range of 30 to 300 mg, the increase in peak plasma concentration and area under the concentration-time curve (AUC) of edaravone exceeded the dose proportion. Edaravone does not accumulate in plasma with once-daily or multiple-dose administration. When edaravone oral suspension is taken with a high-fat meal, its peak plasma concentration (Cmax) and area under the curve (AUC) decrease. In studies in healthy volunteers in Japan and the Caucasus, edaravone was primarily excreted in the urine as a glucuronide conjugate (60-80% of the administered dose within 48 hours). Approximately 6-8% of the dose was excreted in the urine as a sulfate conjugate, and less than 1% was excreted unchanged. In vitro studies showed that edaravone sulfate conjugates hydrolyze back to edaravone, are then converted to glucuronide conjugates in the kidneys, and are ultimately excreted in the urine. After intravenous administration, the mean volume of distribution of edaravone is 63.1 L, indicating its extensive distribution in tissues. After oral administration, the apparent volume of distribution of edaravone is 164 L. Edaravone readily crosses the blood-brain barrier.
The total clearance of edaravone after intravenous administration is estimated to be 35.9 L/h. The apparent total clearance of edaravone after oral administration is estimated to be 67.9 L/h.
Metabolisms/Metabolites
The metabolites of edaravone are not fully understood. Edaravone is metabolized to sulfate conjugates and glucuronide conjugates, neither of which is pharmacologically active. The glucuronide conjugation of edaravone involves multiple uridine diphosphate glucuronyltransferase (UGT) isoenzymes (UGT1A1, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B17). In human plasma, edaravone exists primarily as a sulfate conjugate, presumably generated by sulfonyltransferases. Due to first-pass metabolism, oral edaravone exposure to sulfate and glucuronic acid metabolites was 1.3 times and 1.7 times higher than intravenous edaravone exposure, respectively.

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Biological half-life>
The mean terminal elimination half-life of edaravone is approximately 4.5 to 9 hours.
The half-life of its metabolites is 3 to 6 hours.

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Intravenous administration in rats: The peak plasma concentration (Cmax) was 3.2 μg/mL 5 minutes after administration; the plasma half-life (t1/2) was 1.5 hours [1]
-Due to extensive first-pass metabolism, the oral bioavailability in humans is approximately 3%; after intravenous administration, it rapidly distributes to brain tissue, with a brain/plasma concentration ratio of approximately 0.8 30 minutes after administration [1]
-It is mainly metabolized in the liver via glucuronidation; approximately 70% of the dose is excreted in the urine as glucuronide conjugates within 24 hours [1]

Hepatotoxicity
A small number of patients receiving edaravone treatment may experience elevated serum transaminases, but the frequency, timing, duration, and severity of these elevations are not well understood. The incidence of liver function abnormalities during edaravone treatment is reportedly similar to that during placebo treatment. Most elevations resolve spontaneously, and there are no reports of discontinuation of treatment due to elevated serum enzymes. No clinically significant liver injury caused by edaravone was reported in premarketing trials, nor has it been reported in subsequent clinical applications, although the number of patients treated was small. Therefore, if clinically significant liver injury caused by edaravone does occur, it is certainly very rare. Probability score: E (Unlikely to be the cause of clinically significant liver injury).

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Protein Binding
Edaravone binds to human serum proteins at a rate of 92%, primarily albumin, and this binding is concentration-independent within the range of 0.1 to 50 μmol/L.

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Acute toxicity: LD50 = 103 mg/kg (rat intravenous injection); LD50 = 2200 mg/kg (rat oral administration)[1]
-Subchronic toxicity: Daily intravenous injection of 10 mg/kg in rats for 4 weeks did not cause significant changes in liver and kidney function, hematological parameters or histological abnormalities of major organs[1]
- Human plasma protein binding rate is approximately 94%[1]

[1]. Neuroprotective effects of edaravone: a novel free radical scavenger in cerebrovascular injury. CNS Drug Rev, 2006. 12(1): p. 9-20.

[2]. Edaravone, a free radical scavenger, attenuates cerebral infarction and hemorrhagic infarction in rats with hyperglycemia. Neurol Res, 2013.

[3]. Edaravone reduces astrogliosis and apoptosis in young rats with kaolin-induced hydrocephalus. Childs Nerv Syst. 2016 Dec 17. [Epub ahead of print].

[4]. Protective Effect of Edaravone on Glutamate-Induced Neurotoxicity in Spiral Ganglion Neurons. Neural Plast. 2016;2016:4034218. Epub 2016 Nov 10.

1-Phenylon-3-methyl-5-pyrazolone is a white to off-white powder or crystal. (NTP, 1992)
Edaravone is a pyrazolone compound with the structure 2,4-dihydro-3H-pyrazol-3-one, where the 2 and 5 positions are substituted with phenyl and methyl groups, respectively. It has free radical scavenging and antioxidant effects.
Edaravone is a free radical scavenger and neuroprotective agent with antioxidant properties. It has three tautomers. Edaravone can scavenge reactive oxygen species (ROS), which are closely related to neurological diseases such as amyotrophic lateral sclerosis (ALS) and cerebral ischemia. Intravenous edaravone was first approved in Japan in 2001 for the treatment of acute ischemic stroke. In 2015, edaravone was approved in Japan and South Korea for the treatment of ALS, and subsequently received approval from the US FDA in May 2017 and Health Canada in October 2018, respectively. Edaravone oral suspension received approval from the US FDA in May 2022 and Health Canada in November 2022. On June 19, 2015, edaravone was initially granted orphan drug designation by the European Medicines Agency and underwent regulatory review in Europe. However, due to the Committee on Medicinal Products for Human Use (CHMP) requiring a long-term study to demonstrate the long-term efficacy and safety of edaravone, the manufacturer, Mitsubishi Tanabe Pharmaceutical Co., Ltd., withdrew its Marketing Authorization Application (MAA) for edaravone in the European market on May 24, 2019. Edaravone has also been used to investigate other diseases, such as Alzheimer's disease, neuropathic pain, and ischemic nerve injury. Edaravone is a free radical scavenger and neuroprotective agent used to treat amyotrophic lateral sclerosis (ALS). The incidence of elevated serum transaminases during edaravone treatment is low, but no clinically significant cases of acute liver injury have been found. There are reports and data regarding the use of edaravone in Homo sapiens.
Edaravone is an antipyrine derivative with free radical scavenging and neuroprotective effects. It is used to treat amyotrophic lateral sclerosis (ALS) and stroke.
Drug Indications

Edaravone is approved in the United States and Canada for the treatment of amyotrophic lateral sclerosis (ALS). In Japan, it is also used to treat acute ischemic stroke.
Treatment of Amyotrophic Lateral Sclerosis

Mechanism of Action

Oxidative stress and the production of reactive oxygen species (ROS) are associated with a variety of neurological diseases, such as ALS and cerebral ischemia. Oxidative stress caused by excessive ROS can damage cerebral vascular endothelial cells and neuronal cell membranes, leading to neuronal death. Edaravone is a free radical scavenger that can scavenge and inhibit the generation of hydroxyl radicals and peroxynitrite radicals. The exact mechanism of action of edaravone in ALS has not been fully elucidated; however, edaravone is believed to exert its therapeutic effect through its antioxidant properties.
Since oxidative stress is closely related to the pathophysiological mechanisms of amyotrophic lateral sclerosis (ALS) and cerebral ischemia, inhibiting lipid peroxidation, suppressing lipid peroxide-induced endothelial cell damage, and scavenging free radicals may have neuroprotective effects. Edaravone has no effect on superoxide production. Studies have shown that edaravone may also have anti-inflammatory properties, as it inhibits neutrophil activation and the expression of inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS) in animal models. Studies have also shown that edaravone can alleviate reactive oxygen species (ROS)-induced inflammatory oxidative stress after ischemic cerebral reperfusion.

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Pharmacodynamics
Edaravone delays the progression of neurological diseases such as ischemic stroke and amyotrophic lateral sclerosis (ALS) by limiting the degree of neuronal damage or death.

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Edaravone (MCI-186) is a synthetic free radical scavenger with high selectivity for reactive oxygen species (ROS) and reactive nitrogen species [1, 2, 3, 4].
- Its neuroprotective mechanisms include scavenging ROS, inhibiting lipid peroxidation, reducing oxidative stress-induced neuronal apoptosis, and inhibiting glial cell activation [1, 3, 4].
- Clinically, it is used to treat acute ischemic stroke by reducing neurological deficits by protecting ischemic brain tissue from oxidative damage [1, 2].
- It has also shown potential therapeutic effects in other neurological disorders, such as oxidative stress, hydrocephalus, sensorineural hearing loss (related to spiral ganglion protection), and cerebrovascular injury [3, 4].

C10H10N2O

174.2

174.079

C, 68.95; H, 5.79; N, 16.08; O, 9.18

89-25-8

Edaravone-d5;1228765-67-0

4021

Light yellow to yellow solid

1.2±0.1 g/cm3

333.0±11.0 °C at 760 mmHg

126-128 °C(lit.)

155.2±19.3 °C

0.0±0.7 mmHg at 25°C

1.606

0.44

0

2

1

13

241

0

O=C1CC(C)=NN1C1C=CC=CC=1

QELUYTUMUWHWMC-UHFFFAOYSA-N

InChI=1S/C10H10N2O/c1-8-7-10(13)12(11-8)9-5-3-2-4-6-9/h2-6H,7H2,1H3

5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one

MCI-186; NCI-C03952; MCI 186; NSC 12; MCI186; Radicut; trade name: Radicava; Methylphenylpyrazolone; Norantipyrine; Norphenazone; Phenylmethylpyrazolone; Arone

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 (14.35 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), 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 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (14.35 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (14.35 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.

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