Inhibition of cell death and tissue protection by volatile application of 2,2,6,6-tetramethyl-1-piperidinyloxyl

Abstract

In the present invention, a low-molecular-weight compound 2,2,6,6-tetramtethyl-1-piperidinyloxyl (TEMPO) in the gas state is allowed to act on cells to suppress cell death associated with oxidative stress, thereby protecting tissue from tissue damage during the acute phase of ischemic disease.

Description

[Technical Field]

[0001] The present invention relates to a medicine, and more specifically to an inhibitor of cell death caused by oxidative stress, which is used for the treatment and prevention of cerebral ischemic diseases and the like. [Background technology]

[0002] Oxidative stress-induced cell death is involved in various ischemic disorders, including cerebral infarction. In recent years, there have been dramatic advances in acute treatment (recanalization therapy) for cerebral infarction, where blood vessels in the brain become blocked, and the recanalization rate is steadily increasing. However, brain tissue damage associated with ischemia and reperfusion is unavoidable, and oxidative stress caused by reactive oxygen species is thought to be involved in this. Edaravone, a compound with free radical inhibitory properties, was approved in 2001 as the only neuroprotective drug for use before and after recanalization therapy. Edaravone is also administered intravenously in hospitalized patients to prevent the progression of functional impairment in amyotrophic lateral sclerosis (ALS).

[0003] Regarding cell death accompanied by oxidative stress, iron-dependent cell death, "ferroptosis," has been proposed as a type of oxidative stress-induced cell death that differs from conventional cell death mechanisms such as apoptosis. Ferroptosis, which is carried out by the spread of peroxidation of phospholipids that make up membranes, has been suggested to be involved in neuronal death in the lipid-rich brain. In fact, ferrostatin-1 and liprostatin, which have been developed as ferroptosis inhibitors, have shown cell death-suppressing effects in ischemic experimental models using cultured neurons and brain slices.

[0004] The nitroxyl radical 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO) has the property of capturing lipid radicals generated in biological membranes during the process of ferroptosis (Non-Patent Document 1). It has also been suggested that TEMPO may be useful for protection after recanalization of ischemic diseases and for the prevention or treatment of diseases or conditions associated with free radicals. For example, Patent Document 1 discloses a method for treating cardiovascular disorders such as myocardial infarction using nitroxide compounds such as TEMPO, but does not disclose any treatment targeting brain tissue disorders, such as the treatment of cerebral infarction or ALS. Furthermore, Patent Document 2 discloses a compound in which a nitroxide compound such as TEMPO is stabilized by polymeric micellization using PEG, and a method for using the compound. However, in Patent Documents 1 and 2, the use of TEMPO is mainly in solid or liquid form, such as oral administration or intravenous injection, and there is no mention of the use of TEMPO in volatilized form. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2008-528704 [Patent Document 2] Re-tabled publication 2009 / 133647 [Non-patent literature]

[0006] [Non-Patent Document 1] Griesser et al., J Am Chem Soc 140:3798, 2018 Summary of the Invention [Problem to be solved by the invention]

[0007] Since cell death accompanied by oxidative stress is involved in various ischemic disorders, drugs with antioxidant function may play a role in protecting cells and tissues. For example, the radical scavenger edaravone (MCI-186) is a neuroprotective drug developed in Japan and approved for the first time in the world for use in the acute phase of cerebral infarction. However, the tissue protective effect of edaravone is not necessarily sufficient, and neuroprotective drugs are rarely used in the acute treatment of cerebral infarction in other countries. Furthermore, there are no other cytoprotective drugs other than edaravone for which clinical evidence has been established. The application of ferroptosis inhibitors, ferrostatin-1 and liprostatin, to neuronal cell death has been investigated, but the results are limited to in vitro, and their actual effects are unclear.

[0008] Therefore, an object of the present invention is to provide a drug that is useful for the acute treatment of cerebral ischemic disease and that can rapidly protect tissues against oxidative stress. [Means for solving the problem]

[0009] As a result of extensive research aimed at solving the above problems, the present inventors have discovered that the low molecular weight compound TEMPO acts on living cells in a gaseous state and rapidly suppresses oxidative stress-induced cell death, and have further found that this compound is effective in treating and preventing cerebral ischemic diseases, leading to the completion of the present invention.

[0010] That is, the present invention is as follows. [1] An inhibitor of oxidative stress-induced cell death, comprising: It contains 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO) as an active ingredient. The cell death inhibitor is characterized by acting via a gaseous state. [2] The agent according to [1], wherein the oxidative stress-induced cell death is ferroptosis. [3] The agent according to [1] or [2], wherein TEMPO is partly or entirely composed of gas. [4] The agent according to any one of [1] to [3], which is a liquid or solid preparation, wherein TEMPO is used by volatilizing it. [5] The agent according to any one of [1] to [4], which is for treating or preventing an ischemic disease. [6] The agent according to [5], wherein the ischemic disease is cerebral infarction. [7] The agent according to any one of [1] to [4], which is for treating or preventing amyotrophic lateral sclerosis. [8] The agent according to any one of [1] to [7], which is an inhalant. [9] 1. Use of TEMPO in the manufacture of an inhibitor of oxidative stress-induced cell death, wherein the TEMPO exerts its inhibitory effect on oxidative stress-induced cell death in a gaseous state.

[10] A method for suppressing oxidative stress-induced cell death in a subject, comprising: administering to a subject an effective amount of TEMPO; The method, wherein the TEMPO exerts its inhibitory effect on oxidative stress-induced cell death through its gaseous state. [Effects of the Invention]

[0011] According to the present invention, TEMPO can rapidly reach and penetrate target tissues as a gas, thereby efficiently suppressing cell death due to oxidative stress, particularly ferroptosis, and providing a drug useful for the acute treatment of various ischemic diseases such as cerebral infarction. It is particularly expected to be used in the acute treatment (recanalization therapy) of cerebral infarction, in which cerebral blood vessels are occluded. It is also expected to be used to inhibit the progression of functional impairment in amyotrophic lateral sclerosis (ALS).

[0012] The drug of the present invention can be administered by continuous inhalation and does not require an intravenous route, making it possible to administer it very early to patients with cerebral infarction before they arrive at the hospital, and to administer it to ALS patients at home. Furthermore, since it acts as a gas, it is possible to eliminate the effects of solvents on living organisms, providing a safe drug. [Brief explanation of the drawings]

[0013] [Figure 1] A diagram showing the inhibitory effect of volatile TEMPO on RSL3-induced ferroptosis (in a 10 cm dish). [Figure 2]A diagram showing the inhibitory effect of volatile TEMPO on RSL3-induced ferroptosis (in a 6-well plate). [Figure 3] A diagram showing the inhibitory effect of gaseous TEMPO on RSL3-induced ferroptosis. [Figure 4] A diagram showing the inhibitory effect of TEMPO that was evaporated and then re-eluted on RSL3-induced ferroptosis. [Figure 5] MRI evaluation of cerebral infarction in mice in the TEMPO inhalation administration group and the control group. [Figure 6] A diagram showing the inhibitory effect of inhaled TEMPO on brain tissue damage (some images are cross-sectional images of brain tissue). [Figure 7] A graph comparing survival times between the TEMPO inhalation-administered and control groups in ALS model mice. DETAILED DESCRIPTION OF THE INVENTION

[0014] One aspect of the present invention is an agent for suppressing oxidative stress-induced cell death, comprising: It contains 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO) as an active ingredient. The cell death inhibitor is characterized by acting via a gaseous state.

[0015] [ka] 2,2,6,6-Tetramethyl-1-piperidinyloxyl (TEMPO) is a volatile compound represented by the formula (1). TEMPO is a water-soluble nitroxyl radical with stable radical scavenging properties. Therefore, they are used to suppress cell death associated with oxidative stress, particularly ferroptosis, to protect against tissue damage associated with oxidative stress, and / or to treat or prevent diseases or symptoms associated with oxidative stress.

[0016] Since TEMPO has the ferroptosis-inhibitory effect even when volatilized and applied via a gas, its radical scavenging function and oxidative stress-inhibitory effect are maintained even when applied via a gas. Therefore, the TEMPO contained in the oxidative stress-induced cell death inhibitor of the present invention (hereinafter simply referred to as a cell death inhibitor) may be contained in a solution of any solvent, such as water, DMSO, or a mixture of water and DMSO, and may be volatilized from there, or may be TEMPO volatilized by other means, may be volatilized from solid TEMPO, or may be TEMPO in a gaseous state, but is not limited to these forms, as long as it exhibits ferroptosis inhibition when applied via a gas.

[0017] Oxidative stress-induced cell death is cell death accompanied by oxidative stress. Oxidative stress caused by reactive oxygen species is believed to be involved in this process. Oxidative stress-induced cell death is believed to be involved in tissue damage associated with ischemia and reperfusion. Suppression of oxidative stress-induced cell death can be achieved, for example, by antioxidant activity, radical scavenging function, or free radical prevention activity. Substances having the above-mentioned action or function include TEMPO. Therefore, substances that achieve the inhibition of oxidative stress-induced cell death include TEMPO. Oxidative stress-induced cell death includes ferroptosis. Oxidative stress is also believed to be involved in a neuronal cell death model in which glutamate is administered to mouse hippocampal cells, and volatilized TEMPO inhibits cell death in this neuronal cell death model as well.

[0018] Ferroptosis is an oxidative stress-induced cell death caused by increased reactive oxygen species and lipid peroxidation, which are generated depending on intracellular free iron. Ferroptosis is carried out by the spread of peroxidation of phospholipids that constitute membranes, and is thought to be particularly involved in the death of neurons in the lipid-rich brain. However, the ferroptosis inhibited in the present invention is not limited to specific tissues. Ferroptosis can be artificially induced by treating cancer cells with Ras mutations, such as HT1080 cells (human invasive fibrosarcoma), CALU-1 cells (human lung epidermoid carcinoma), and PANC-1 cells (human pancreatic adenocarcinoma), with drugs such as erastin and RSL3.

[0019] Specifically, the suppression of oxidative stress-induced cell death may mean a reduction in the rate of cell death due to oxidative stress. The rate of cell death is expressed, for example, by the following formula (2): Cell death rate (%) = dead cells / (live cells + dead cells) × 100 (2) Suppression of oxidative stress-induced cell death is achieved, for example, by antioxidant activity, radical scavenging function, or free radical inhibitory activity. An agent that achieves the inhibition of oxidative stress-induced cell death is called an inhibitor of oxidative stress-induced cell death. In the present disclosure, the cell death inhibitor particularly refers to an inhibitor of oxidative stress-induced cell death.

[0020] Acting via a gaseous state means, for example, that a volatilized cell death inhibitor or a cell death inhibitor that is partially or completely gaseous may act directly on cells, tissues, or a living organism, or that a volatilized cell death inhibitor or a cell death inhibitor that is partially or completely gaseous may act on cells, tissues, or a living organism after dissolving in any bodily fluid such as blood, tissue fluid, or cerebrospinal fluid.

[0021] The present invention relates to an agent for treating or preventing diseases that can be treated or prevented by suppressing cell death due to oxidative stress. Such diseases include ischemic diseases. Ischemic diseases include ischemic brain disease, ischemic heart disease, ischemic kidney disease, etc. Ischemic brain disease includes cerebral infarction, and ischemic heart disease includes myocardial infarction, but is not limited to these.

[0022] In ischemic diseases, vascular recanalization is performed for acute treatment of ischemia. In this case, oxidative stress caused by reactive oxygen species and oxidative stress-induced cell death, particularly ferroptosis, are thought to be involved in tissue damage caused by ischemia and reperfusion. TEMPO contained in the agent of the present invention scavenges reactive oxygen species and exhibits inhibitory effects on oxidative stress-induced cell death, particularly ferroptosis, and is therefore effective in treating or preventing tissue damage in ischemic diseases. For example, administration of gaseous TEMPO to mice subjected to permanent middle cerebral artery occlusion (MCAO) provides brain tissue protection. MCAO is a surgical stroke model used to examine the effects of drug administration after the onset of cerebral infarction.

[0023] Another disease involving cell death due to oxidative stress is amyotrophic lateral sclerosis (ALS). ALS is a progressive disease in which motor neurons degenerate and / or are lost. ALS is thought to be caused by mutations in genes encoding enzymes involved in the decomposition of reactive oxygen species, the involvement of free radicals, or neuronal damage due to glutamate toxicity. TEMPO contained in the agent of the present invention scavenges reactive oxygen species to suppress oxidative stress-induced cell death, and also suppresses glutamate toxicity in a neuronal cell death model in which glutamate is administered to mouse hippocampal cells. Therefore, it is expected to be effective in treating or preventing ALS.

[0024] The dosage form of the cell death inhibitor of the present invention is not particularly limited as long as TEMPO acts as a gas during use, and may be a solid formulation, a liquid formulation, a volatile solid or liquid formulation, a formulation containing a gaseous portion, a formulation containing a gas and a solution, or a formulation composed of a gas, but is not limited to these forms. The TEMPO formulation may be a formulation that is dispensed in an appropriate amount upon use, or may be a formulation in which an appropriate amount of TEMPO is dispensed in advance into a vial or the like. The cell death inhibitor is preferably in the form of a liquid preparation in which TEMPO is dissolved in a solvent. The solvent is not particularly limited as long as it can dissolve TEMPO, but examples include water, organic solvents such as dimethyl sulfoxide (DMSO) or ethanol, or mixtures of such organic solvents with water. In particular, a solution in which the solvent is water, DMSO, or a mixture of water and DMSO is more preferred. Another embodiment of the present invention is a liquid formulation in which water, DMSO, or the like is used as a solvent, and the concentration of TEMPO is 10 μM to 100,000 μM when water is used as the solvent, or 10 μM to 500,000 μM when DMSO is used as the solvent. When a liquid formulation is used, for example, TEMPO vaporized from the liquid formulation may be used as is, or the vaporized TEMPO may be recovered and used. In this case, the conditions for volatilizing TEMPO are not particularly limited, and may be at room temperature and atmospheric pressure, or may be heated.

[0025] A solid formulation containing solid TEMPO is also preferred as a dosage form of the cell death inhibitor of the present invention. The form of the solid formulation is not particularly limited, but may be, for example, powder. Because solid TEMPO can volatilize naturally, the solid formulation may be used, for example, by using TEMPO vaporized from the solid formulation as is, or by using gas recovered from the vaporized TEMPO. In this case, the conditions for volatilizing TEMPO are not particularly limited, but may be room temperature and atmospheric pressure, or may be heated. Furthermore, solid formulations are expected to be stable during storage, and can be stored sealed in a cool, dark place.

[0026] The cell death inhibitor according to the present invention is an agent containing TEMPO, and may contain other pharmaceutically acceptable ingredients. The other ingredients are not particularly limited and may be any ingredients used in pharmaceuticals, such as buffers. In particular, in embodiments containing liquid formulations, buffers can contribute to stability during storage and prevention of changes in properties during use. Pharmaceutically acceptable buffers typically include citrate buffers or phosphate buffers, which include citric acid, sodium citrate, and mixtures thereof, and phosphate buffers which include phosphoric acid, monosodium phosphate, disodium hydrogen phosphate, and mixtures thereof. Furthermore, the cell death inhibitor according to the present invention, particularly in an embodiment including a liquid formulation, may contain a diluent or may be diluted with a diluent. Typical diluents include water, DMSO, and the like. From the viewpoint of toxicity to cells, tissues, and living organisms, water is preferred as the diluent.

[0027] The dosage of the cell death inhibitor according to the present invention can be appropriately determined depending on each embodiment. For example, in the case of a liquid preparation using water or DMSO as a solvent as described above as one embodiment, the dosage is within the above-mentioned concentration range and is an amount that can be evaporated at 4°C to 100°C, preferably an amount that can be evaporated when heated to 23°C to 70°C, and more preferably an amount that can be evaporated when heated to 23°C to 40°C. The cell death inhibitor of the present invention may be administered once or several times as needed, and may be administered continuously for a required period at a frequency of once a day, 2 to 4 times a day, 2 to 4 times a week, once a week, or once every two weeks, or may be administered continuously for a period of several minutes to several hours.

[0028] The method of administering the cell death inhibitor of the present invention to a subject such as a human is not particularly limited as long as it is a method in which TEMPO is administered to a subject such as a human as a gas, and it can be administered in a gaseous state using the gaseous portion of the agent or a vaporized agent. Specifically, it can be administered by, for example, directly inhaling the gas, allowing it to volatilize naturally and then inhaling, impregnating it with absorbent cotton and then inhaling, heating it and then inhaling it, or volatilizing it with a vaporizer and then inhaling it, or it can be administered by a combination of these methods, for example, impregnating it with absorbent cotton and then heating it and then inhaling it. Furthermore, particularly in embodiments involving liquid formulations, it may be administered after diluting it with a diluent to an appropriate concentration as needed.

[0029] TEMPO may be used in combination with other medications, such as edaravone (MCI-186) and vitamin E. [Example]

[0030] The present invention will be described below using examples, but the present invention is not limited to these examples.

[0031] [Example 1] Inhibition of ferroptosis by volatile TEMPO Using the following method, we applied TEMPO in a gaseous state to cells in which ferroptosis had been induced, and confirmed its effects. Human invasive fibrosarcoma HT1080 cells (1 × 10 5 ) were seeded onto 3.5 cm dishes and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin at 37°C under 5% CO2 for 40 hours. Another 3.5 cm dish containing 1 mL of distilled water was placed next to the 3.5 cm dish containing the cells in the 10 cm dish. The HT1080 cell culture medium was replaced with RSL3-supplemented medium (RSL3 (Selleck) dissolved in dimethyl sulfoxide to 1 mM, 1 / 1000 of the medium volume). At the same time, TEMPO was added to the distilled water to final concentrations of 10, 100, or 1000 μM. TEMPO was dissolved in DMSO to a final concentration of 100 mM. As a control for the inhibitory effect, Trolox, a vitamin E derivative, was added to the cell culture medium at a final concentration of 0.2 mM. After 5 hours, all cells were harvested and stained with trypan blue solution to count the number of live and dead cells. The cell death rate was calculated using the following formula (2), and the results are shown in Figure 1 as the mean ± standard error. Cell death rate (%) = dead cells / (live cells + dead cells) × 100 (2)

[0032] TEMPO significantly inhibited RSL3-induced ferroptosis at concentrations above 10 μM, and significantly inhibited RSL3-induced ferroptosis at concentrations above 100 μM. Similar results were obtained when erastin (10 μM), another ferroptosis-inducing agent, was used instead of RSL3, and when different cancer cell lines, CaluI and PancI, were used. Similar results were also obtained in a neuronal cell death model using glutamate-treated mouse hippocampal HT22 cells. In this experiment, TEMPO added to water did not directly mix with the cell culture medium, so it is likely that it acted on the cells via the gas after evaporation. Therefore, it was confirmed that volatilized TEMPO inhibits ferroptosis via its gaseous state.

[0033] [Example 2] Inhibition of ferroptosis by volatile TEMPO HT1080 cells were seeded in the upper left well of a 6-well plate in the same manner as in Example 1 and cultured for 40 hours. The culture medium was replaced with RSL3-supplemented medium, and at the same time, 1 μL of TEMPO (100 mM) solution was added to the lower right well of the plate. The ferroptosis inhibitory effect was measured using the same method as in Example 1. The results are shown in Figure 2 as the mean ± standard error. As a result, the cell death rate decreased, and RSL3-induced ferroptosis was inhibited. Therefore, ferroptosis was inhibited by TEMPO volatilizing from the DMSO solution approximately 10 cm away and acting in a gaseous state.

[0034] Example 3: Inhibition of ferroptosis by gaseous TEMPO Air and 1 μL of TEMPO (100 mM) were added to an aluminum gas bag and left in an incubator at 37°C for 3 hours. The gas (20 mL) inside the gas bag was collected using a gas-tight syringe and gently injected into a 10 cm dish containing RSL3-treated HT1080 cells from a 3.5 cm dish. The RSL3-treated HT1080 cells were obtained using the same method as in Example 1. The ferroptosis inhibitory effect was then measured using the same method as in Example 1. The results are shown in Figure 3 as mean ± standard error. As a result, the cell death rate decreased, indicating that RSL3-induced ferroptosis was inhibited. Therefore, it is believed that ferroptosis was inhibited by the presence of the gas component (gas) of TEMPO released by heating.

[0035] [Example 4] Inhibition of ferroptosis by TEMPO volatilized and re-eluted A 3.5 cm dish containing 1 mL of distilled water and another 3.5 cm dish containing 1 μL of TEMPO (100 mM) were placed side by side in a 10 cm dish and left in an incubator at 37°C. After 3 hours, 10 μL of distilled water was collected and added to RSL3-treated HT1080 cells in a separately prepared 3.5 cm dish. The RSL3-treated HT1080 cells were obtained using the same method as in Example 1. The ferroptosis inhibitory effect was then measured using the same method as in Example 1. The results are shown in Figure 4 as mean ± standard error. As a result, the cell death rate decreased, and RSL3-induced ferroptosis was inhibited. Therefore, it is believed that the volatilized TEMPO gas was re-eluted into the distilled water, and that the re-eluted TEMPO inhibited ferroptosis.

[0036] Example 5: Suppression of brain tissue damage in a mouse model of cerebral infarction by volatile TEMPO The gas-mediated effects of volatile TEMPO were confirmed using a mouse model of cerebral infarction caused by permanent middle cerebral artery occlusion (MCAO) using the following method. For comparison, edaravone (MCI-186; Mitsubishi Tanabe Pharma), a compound with free radical scavenging properties, was used. This is the only neuroprotective drug approved in 2001 that provides neuroprotection before and after recanalization therapy.

[0037] Male mice (CB-17 / Icr- + / + Jcl; CLEA Japan) aged 7-10 weeks (weight 30-35 g) were used. Mice were housed 2-3 per cage and maintained in a room with a temperature of 24±1°C and humidity of 55±5% under a 14-hour / 10-hour light / dark cycle for at least 2 days prior to surgery. Food and water were available ad libitum before and after surgery. The MCAO model was performed using the 1981 Tamura rat model (J Cereb Blood Flow Metab 1:53, 1981) with minor modifications. Mice were anesthetized with isoflurane and placed on a feedback heating pad, with the rectal core temperature maintained at 37.0°C ± 0.5°C. The base of the zygomatic bone was removed using a small dental drill and steel burr to expose the base of the temporal bone. A small hole measuring approximately 4 x 4 mm was then drilled with a diamond burr to expose the middle cerebral artery. After cutting the dura, electrocoagulation was performed using a 1-mm-tipped animal bipolar forceps (Jewelar Bipolar Forceps). After visual confirmation of blood flow occlusion, the cauterized vessels were cut, and the wound was cleaned and sutured. Rectal temperature was carefully monitored during recovery from anesthesia.

[0038] Fifteen minutes after the creation of the infarction, each mouse was placed individually in a sealed cage measuring 17 × 10 × 10 cm with an air hole and randomly assigned to one of the following three groups. For the control group, a 3.5 cm dish containing 0.1 g of cotton and soaked in 5 ml of PBS heated to 60°C was placed in the center of the cage. For the TEMPO inhalation group, 0.1 g of cotton was placed in a 3.5 cm dish, soaked in 5 ml of 100 mM TEMPO solution heated to 60°C, and placed in the center of the cage. The edaravone group received 0.1 ml of edaravone adjusted to 3 mg / ml intravenously and was then returned to their cages. For each mouse, T2-weighted MRI images were taken 3, 5, and 7 hours after infarction. The signal intensity ratio of the MRI images was calculated as the ratio (average infarcted area / average non-infarcted area) of the average measured values ​​of three points located at a fixed distance from the infarcted area and three points on the non-infarcted side that were symmetrical to each other. The ratios calculated from the images taken 3, 5, and 7 hours after infarction are shown in Figure 5.

[0039] Eight hours later, the animals were observed for neurological symptoms (limb paralysis, motor ability, and feeding disorders) and then sacrificed. Brain tissue sections were stained with 2,3,5-triphenyltetraolium chloride (TTC). Unstained areas were considered infarct lesions, and the infarct area was measured using ImageJ software. The cerebral infarct volume was calculated by multiplying the infarct area by the slice thickness (1 mm). Figure 6 shows photographs of TTC-stained brain tissue sections from the control group and the TEMPO inhalation group, as well as the mean values ​​± standard error of the cerebral infarct volumes from the control group, edaravone-administered group, and TEMPO inhalation-administered group.

[0040] As a result, brain tissue damage was more strongly suppressed in mice receiving TEMPO by natural inhalation than in control or edaravone-treated mice. Furthermore, when the concentration of TEMPO added to the 3.5 cm dish was examined, the protective effect was identical at 25 mM, while the effect was weaker at 12.5 mM.

[0041] Example 6: Volatile TEMPO suppresses muscle atrophy in ALS model mice The effects of volatile TEMPO were confirmed using ALS model mice (C57BL / 6-hSOD1(G93A)). Transgenic mice carrying a mutant Cu / Zn superoxide dismutase (SOD1:G93A) gene are widely used as a useful model of amyotrophic lateral sclerosis (ALS). Symptoms appear around 90 days after birth, and muscle atrophy progresses within two months, leading to death.

[0042] Male mice (C57BL / 6-hSOD1(G93A)) aged 14-15 weeks (weight 24-30 g) were used. Mice were housed individually in cages in a room set at 24±1°C and 55±5% humidity, with a 14-hour / 10-hour light / dark cycle. Food and water were available ad libitum. Mice of approximately equal weight were divided into two groups of five for each experiment. For the control and TEMPO treatments, 5 ml of sterile water or TEMPO solution (0.1 M) was soaked in 5 ml of polystyrene tubes with approximately 10 nail holes, containing 0.1 g of cotton. The tubes were then placed in the cage. The sterile water and TEMPO solution were changed every 84 hours until the mice died.

[0043] The number of days that experimental mice survived from the start of TEMPO (control) treatment until death was counted, and the results were plotted and expressed as the mean ± standard error (Figure 7). As a result, the survival period was extended in mice that received TEMPO by natural inhalation compared to the control.

Claims

1. An inhibitor of cell death caused by oxidative stress, It contains 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO) as an active ingredient, A cell death inhibitor characterized by acting via a gaseous state.

2. The cell death inhibitor according to claim 1 , wherein the cell death caused by oxidative stress is ferroptosis.

3. The cell death inhibitor according to claim 1 or 2, wherein TEMPO is partly or entirely composed of gas.

4. The cell death inhibitor according to any one of claims 1 to 3, which is a liquid or solid formulation and is used by volatilizing TEMPO.

5. The cell death inhibitor according to any one of claims 1 to 4, which is used for treating or preventing an ischemic disease.

6. The cell death inhibitor according to claim 5, wherein the ischemic disease is cerebral infarction.

7. The cell death inhibitor according to any one of claims 1 to 4, which is for treating or preventing amyotrophic lateral sclerosis.

8. The cell death inhibitor according to any one of claims 1 to 7, which is an inhalant.

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