JP2005213173A - Astroglia cell death inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a compound which can minimally stop the death of astroglia cells due to hydrogen peroxide to maintain the absolute number of the cells at a high level and can thereby maximally withdraw the functions of nerve cells to prevent or treat nervous diseases such as Alzheimer's disease. <P>SOLUTION: This astroglia cell death inhibitor is characterized by containing a compound represented by formula (I) (R<SP>1</SP>is methyl or ethyl; R<SP>2</SP>is H or a 1 to 5C straight chain or branched alkyl; R<SP>1</SP>and R<SP>2</SP>may be identical or different from each other) or its salt. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a cell death inhibitor of astroglial cells, and in particular, is a compound that acts in a suppressive direction on cell death of astroglial cells, and treatment or prevention of Alzheimer's disease or Parkinson's disease, or brain function The present invention relates to a cell death inhibitor that can be used as an improving agent.

At present, few drugs and compounds that improve nervous system diseases such as Alzheimer's disease and Parkinson's disease are known. While such definitive therapeutic agents are scarce, acetylcholinesterase inhibitors have attracted attention. This medicine has the function of degrading the neurotransmitter acetylcholine and inhibiting the enzyme that blocks transmission (acetylcholinesterase), and this action is expected to improve the symptoms of Alzheimer's disease. (For example, refer to Patent Documents 1 to 3).
JP-A-8-268949 JP-T 6-510788 JP-A-9-268176

However, as elucidation of neurological diseases such as Alzheimer's disease progresses, it has been found that astroglial cells are deeply involved as another onset mechanism, and it is not possible to cure neurological diseases or improve symptoms from different perspectives It has been proposed. That is, there is no doubt that nerve cells are the main players working in the brain, but there are about nine times as many glial cells in the brain as nerve cells, which help nerve cells. In particular, astroglia cells not only exist as cells that support the structure of the brain, but also play a role in regulating neurotransmission and regulating brain damage. In other words, nerve cells appear to play the leading role in the brain, but do not function properly without the support of astroglial cells. It has recently been found that astroglial cells also induce cell death by hydrogen peroxide (H ₂ O ₂ ) produced in the body and the number of cells decreases. When astroglial cells are exposed to hydrogen peroxide produced in the body, the glial cells are induced in the direction of cell death.

  Therefore, the present inventor has focused on the suppression of astroglial cell death in the treatment or prevention of nervous system diseases such as Alzheimer's disease. That is, by preventing the death of astroglial cells by hydrogen peroxide to a minimum and maintaining the absolute number at a high level, it is possible to prevent or cure a nervous system disease caused by a decrease in the number of astroglial cells. Therefore, various compounds having such an effect (which can avoid (prevent) astroglial cell death even when exposed to hydrogen peroxide) have been tried. As a result of several trials and errors, the present inventors finally found out that certain compounds suppress the death of astroglial cells, and led to the present invention.

The cell death inhibitor of astroglial cells according to claim 1 is a compound that suppresses cell death of astroglial cells, and comprises a compound represented by the following general formula (I) or a salt thereof: It is.

[In the general formula (I), R ¹ is a methyl group or an ethyl group, R ² is a hydrogen atom or a linear or side chain alkyl group having 1 to 5 carbon atoms, and R ¹ and R ² are the same. Or it may be different].

  The cell death inhibitor according to claim 2 is the cell death inhibitor according to claim 1, wherein the compound is 2-methoxyphenol, 2-methoxy-4-methylphenol, and 2-methoxy-4. -At least one selected from the group consisting of ethylphenol.

  The cell death inhibitor according to claim 3 is the cell death inhibitor according to claim 1 or 2, and is an anti-dementia drug (including Alzheimer's disease), a Parkinson's disease therapeutic drug, a cerebrovascular disorder therapeutic drug or It is for brain function improving medicine.

  According to the present invention, killing of astroglial cells by hydrogen peroxide (decrease in the number of astroglial cells) can be minimized, so that the function of nerve cells supported by the astroglial cells can also be maintained. In addition, it can treat or prevent nervous system diseases such as Alzheimer's disease, and can be used more effectively by combining with other drugs having another mechanism (for example, the above-mentioned acetylcholinesterase inhibitors). Can be prevented or treated.

Astroglial cell death inhibitor As described above, the astroglial cell death inhibitor of the present invention comprises a compound represented by the following general formula (I) or a salt thereof.

R ¹ in the general formula (I) is a methyl group or an ethyl group, R ² is a hydrogen atom or a linear or side chain alkyl group having 1 to 5 carbon atoms, and R ¹ and R ² are the same. May be different. Specific examples will be given below. In this illustration, “butyl” is a generic term for n-butyl, isobutyl, s-butyl and t-butyl, and “propyl” is a generic term for n-propyl and isopropyl. It is a thing. That is, 2-methoxyphenol (guaiacol), 2-methoxy-3-methylphenol, 2-methoxy-3-ethylphenol, 2-methoxy-3-propylphenol, 2-methoxy-3-butylphenol, 2-methoxy-4 -Methylphenol, 2-methoxy-4-ethylphenol, 2-methoxy-4-propylphenol, 2-methoxy-4-butylphenol, 2-methoxy-5-methylphenol, 2-methoxy-5-ethylphenol, 2- Methoxy-5-propylphenol, 2-methoxy-5-butylphenol, 2-ethoxyphenol, 2-ethoxy-3-methylphenol, 2-ethoxy-3-ethylphenol, 2-ethoxy-3-propylphenol, 2-ethoxy -3-butylphenol, 2-eth Ci-4-methylphenol, 2-ethoxy-4-ethylphenol, 2-ethoxy-4-propylphenol, 2-ethoxy-4-butylphenol, 2-ethoxy-5-methylphenol, 2-ethoxy-5-ethylphenol 2-ethoxy-5-propylphenol, 2-ethoxy-5-butylphenol and the like.

Among them, R ² having 1 to 3 carbon atoms is preferable in terms of excellent ability to prevent astroglial cell death, and more preferably a methyl group or an ethyl group. R ² is preferably bonded to the 3 or 4 position from the viewpoint of excellent ability to prevent the death of astroglial cells, more preferably bonded to the 4 position, Guayacol, 2-methoxy-4-methylphenol (hereinafter also simply referred to as “2M4MP”), 2-methoxy-4-ethylphenol (hereinafter simply referred to as “2”) is superior in the ability to prevent the death of astroglial cells. 2M4EP ”, 2-ethoxy-4-methylphenol (hereinafter also simply referred to as“ 2E4MP ”), 2-ethoxy-4-ethylphenol (hereinafter also simply referred to as“ 2E4EP ”), and among them, guaiacol, 2M4MP and 2M4EP are most preferred. In addition, the compound shown by the said general formula (I) may be used individually by 1 type, and can also use 2 or more types together.

These guaiacol, 2M4MP, and 2M4EP are all components of wood creosote (Ogata N., Baba T. Analysis of beechwood creosote by gas chromatography-mass spectrometry and high-performance liquid chromatography. Res Commun Chem Pathol Pharmacol 66, 411-423 (1989), analysis of beech wood creosote by gas chromatography / mass spectrometry and high performance liquid chromatography). N. Ogata et al., Pharmacology, 46, (1993), p. 173, describes that creosote has an antipruritic action based on inhibition of intestinal motility. In addition, it is listed as a disinfectant in the category of antidiarrheal drugs in column V in the gastrointestinal drug production approval standards of the 1988 edition, page 240, edited by the Pharmaceutical Production Guidelines (Japan Standards Association). In addition, Hiroshi Ito's "Pharmacology" (Korodo Co., Ltd., published 6th edition, revised on January 5, 1983), page 416, wood creosote is used for intestinal preservatives, and is also an expectorant effect by inhalation indication. In the Japanese Pharmacopoeia, it is described that it is used for expectoration, intestinal abnormal fermentation, food poisoning, and the like. The United States Dispensatory, 27th ed. (1973), page 355 also states that wood creosote is used as a fungicide for external use and as an expectorant for internal use. ing.

Salt Form The cell death inhibitor of the present invention can also be made into a salt form using a base. The salt to be used is not particularly limited as long as it is a pharmaceutically (pharmaceutically) acceptable salt. For example, metal salts such as sodium, calcium, potassium, magnesium, lithium, and amines (monoethanolamine) , Diethanolamine, triethanolamine) and ammonium salts.

Nervous system disease Examples of the target disease name of the astroglial cell death inhibitor of the present invention include dementia such as Alzheimer's disease, Parkinson's disease and depression. Furthermore, it can be expected to be applied to improve the function after cerebrovascular disorder or cerebral ischemia and to improve (promote) the rehabilitation effect.

Amount used The dose (use amount) of the astroglial cell death inhibitor of the present invention varies depending on the type or sex of the target animal, age, and symptom level. In the case of administration or rectal administration (suppository), it is about 0.1 to 10 mg, preferably 0.5 to 5 mg per 1 kg of adult body weight per day. In this case, it is 0.05 to 5 mg, preferably 0.25 to 2.5 mg, per 1 kg of adult body weight per day. These daily doses can be administered once or divided into 2 to 4 minutes or more.

Dosage Form The astroglial cell death inhibitor of the present invention is used in a general form in medical drugs. Common forms include, for example, tablets, pills, powders, capsules (soft capsules, hard capsules), granules, troches, chewables, internal liquids, and injections (intravascular administration, intramuscular) Administration, subcutaneous administration, intradermal administration, etc.), or suppositories. In preparing tablets, granules, powders, conventionally known carriers can be widely used. For example, excipients such as lactose, sucrose, glucose, starch, crystalline cellulose, such as hydroxypropylcellulose, methylcellulose, Binders such as gelatin, tragacanth, gum arabic, and sodium alginate, for example, disintegrating agents such as starch, carboxymethylcellulose, and calcium carbonate, and lubricants such as magnesium stearate, talc, and stearic acid can be used. If necessary, the tablet can be given a normal coating, for example, a sugar-coated tablet, a film-coated tablet, or the like, and may be a bilayer tablet or a multilayer tablet. Granules and powders can also be given a normal coating.

  The astroglial cell death inhibitor of the present invention can be taken not only as a pharmaceutical product but also as a general food or drink. That is, confectionery (cookies, biscuits, cakes, buns, snacks, gums, candy, etc.) and drinking water (nutrition drinks, carbonated drinks, lactic acid drinks, soft drinks, etc.), instant foods (instant noodles, instant curry, Stew, etc.), kneaded food (ham, sausage, kamaboko, etc.), processed oils (butter, margarine, mayonnaise, dressing, etc.), seasonings (sauce, soy sauce, salt, pepper, etc.), dairy products (yogurt, processed milk, etc. ) Can be taken in the astroglial cell death inhibitor of the present invention.

  An embodiment of the present invention will be described below, but the present invention is not limited thereby.

Culture of astroglial cells (astrocytes) (see Fig. 1)
Astroglia cells were cultured by a conventionally known method. That is, cerebral cortex (cortex) was taken out from a Wistar rat 20-day-old fetus. This was treated with 0.25% trypsin solution at 37 ° C. for 20 minutes. This was implanted into a culture dish coated with polyethyleneimine and cultured in DMEM (Dulbecco's modified MEM) containing 10% fetal calf serum (FCS). One week after culturing, the cells were detached with 0.25% trypsin solution and seeded again on the plate. Further, after 1-2 weeks of culture, the cells were detached with a 0.25% trypsin solution, seeded on a 96-well microplate, and used for the experiment.

  In addition, as shown in FIG. 2 below, as an identification test, the glial fibrillary acidic protein (GFAP) and the microtubule-associated protein (MAP-2) were analyzed by the immunocytochemistry method and the Western blotting method. As a result of examining the positive / negative reaction, it was determined that astroglial cells were cultured instead of neurons.

Effect of astroglial cells by hydrogen peroxide (see Fig. 3)
Astroglial cells were seeded in a 96-well microplate with 100 μg of culture solution so that the number of cells in each well was about 1.2 × 10 ⁵ cells / cm ^3, and cultured in an incubator for 24 hours (5% (37 ° C. under carbon dioxide atmosphere). Thereafter, 10 μl of hydrogen peroxide (H ₂ O ₂ ) prepared to a final concentration (0 μM, 30 μM, 100 μM, 300 μM, and 1 mM) is added to each well to expose astroglial cells to hydrogen peroxide. did. After 2 hours of exposure, the medium was changed in the same amount, and a solution of Cell Counting Kit-8 (manufactured by Dojin Chemical Co., Ltd., Japanese Patent Publication No. 2757348), which is one of the reagents for measuring cell proliferation / cytotoxicity (2- ( 2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium monosodium salt (WST-8) 5mmol / L, 1-methoxy phenazine methosulfate (1-methoxy PMS) 0.2 mmol / liter, NaCl 150 mmol / liter) was added to each well. Then, a color reaction was carried out in a carbon dioxide incubator for 30 minutes, and the absorbance at 450 nm (reference wavelength 650 nm) was measured using a microplate reader.

  As a result, as shown in FIG. 4, when hydrogen peroxide prepared at 30 μM was used, cell death was confirmed to some extent, but was insufficient, and at 100 μM, 300 μM and 1 mM, cell death was remarkable. Therefore, in the subsequent experiments, the concentration of hydrogen peroxide that induces cell death of astroglial cells was set to 100 μM.

Example 1 (Induction of cell death by hydrogen peroxide and inhibition of cell death by guaiacol)
For astroglial cells, guaiacol (0 μM, 10 μM, 100 μM, 1000 μM) and hydrogen peroxide (concentration 100 μM) were exposed to the cells for 2 hours. The survival rate of astroglial cells after 2 hours was compared to a system that was not exposed to hydrogen peroxide (concentration 100 μM). The results are shown in FIG. 5 and FIG.

  As is apparent from the figure, cell death of astroglial cells is caused by 100 μM hydrogen peroxide (guaiacol concentration 0 μM), but in a system using hydrogen peroxide and guaiacol (concentrations 10 μM, 100 μM, 1000 μM) in combination, As the survival rate of astroglial cells has increased, it is considered that guaiacol has prevented the induction of cell death by hydrogen peroxide, and guaiacol has the effect of preventing cell death of astroglial cells by hydrogen peroxide. This is clear. This is particularly noticeable when the concentration is 1 mM.

Example 2 (Induction of cell death by hydrogen peroxide and inhibition of cell death by 2M4MP)
For astroglial cells, 2M4MP (0 μM, 10 μM, 100 μM, 1000 μM) and hydrogen peroxide (concentration 100 μM) were exposed to the cells for 2 hours. The survival rate of astroglial cells after 2 hours was compared to a system that was not exposed to hydrogen peroxide (concentration 100 μM). The results are shown in FIG. 5 and FIG.

  As is apparent from the figure, cell death of astroglial cells is brought about by 100 μM hydrogen peroxide (2M4MP concentration 0 μM), but in a system using hydrogen peroxide and 2M4MP (concentrations 10 μM, 100 μM, 1000 μM) in combination, Since the survival rate of astroglial cells has increased, it is considered that 2M4MP has prevented the induction of hydrogen peroxide cell death, and 2M4MP has the effect of preventing cell death of astroglial cells by hydrogen peroxide. This is clear. This is particularly noticeable when the concentration is 100 μM.

Example 3 (Induction of cell death by hydrogen peroxide and inhibition of cell death by 2M4EP)
For astroglial cells, 2M4EP (0 μM, 10 μM, 100 μM, 1000 μM) and hydrogen peroxide (concentration 100 μM) were exposed to the cells for 2 hours. The survival rate of astroglial cells after 2 hours was compared to a system that was not exposed to hydrogen peroxide (concentration 100 μM). The results are shown in FIG. 5 and FIG.

  As is apparent from the figure, cell death of astroglial cells is brought about by 100 μM hydrogen peroxide (2M4EP concentration 0 μM), but in a system using hydrogen peroxide and 2M4EP (concentration 10 μM, 100 μM, 1000 μM) in combination, Since the survival rate of astroglial cells has increased, it is considered that 2M4EP has blocked the induction of hydrogen peroxide cell death. 2M4EP has the effect of preventing cell death of astroglial cells by hydrogen peroxide. This is clear. This is particularly noticeable when the concentration is 100 μM to 1 mM.

Formulation Example 1 (pills)
(Prescription 1)
Guayacol, 2M4MP or 2M4EP 5mg
Daylily 25mg
Glycerin 10mg
Ordinary water 50mg
Each component of the following formulation is kneaded, the pill lump is divided by a rounding machine, and rounded by a rounding machine, and a round containing 5 mg of the astroglial cell death inhibitor of the present invention in one round. An agent was prepared.

Formulation Example 2 (Hard capsule)
(Prescription 2)
Guayacol, 2M4MP or 2M4EP 10mg
Starch 250mg
Guayacol, 2M4MP or 2M4EP and starch were mixed to obtain a mixed powder, and filled into a hard capsule to prepare a hard capsule containing 10 mg of the astroglial cell death inhibitor of the present invention in one capsule.

Formulation Example 3 (Soft capsule)
(Prescription 3)
Guayacol, 2M4MP or 2M4EP 10mg
Olive oil 200mg
A solution was obtained by dissolving in guaiacol, 2M4MP or 2M4EP and olive oil, filled in a soft capsule, and a soft capsule containing 10 mg of the astroglial cell death inhibitor of the present invention in one capsule was prepared.

Formulation Example 4 (tablet)
(Prescription 4)
Guayacol, 2M4MP or 2M4EP 20mg
Lactose 250mg
Methylcellulose 3mg
Magnesium stearate 2mg
Carboxymethylcellulose 10mg
The ingredients other than magnesium stearate of the above formulation are mixed and kneaded with water to form granules. After drying, the granules are mixed with magnesium stearate and compression molded, or the ingredients of the above recipe are mixed. Then, it was directly compression-molded to prepare 1 tablet of 285 mg.

  According to the present invention, killing of astroglial cells by hydrogen peroxide (decrease in the number of astroglial cells) can be minimized, so that the function of nerve cells supported by the astroglial cells can also be maintained. Can treat or prevent neurological diseases such as dementia (including Alzheimer's disease treatment), Parkinson's disease, or can be applied to improve brain function, and other drugs with other mechanisms (eg, The acetylcholinesterase inhibitor) can be used in combination to prevent or treat the disease more effectively.

It is the schematic explanatory drawing which showed the culture method of the astroglia cell. It is the schematic explanatory drawing which showed the procedure which adds a hydrogen peroxide to the cultured astroglia cell and induces cell death. It is the figure of the microscope picture which showed that GFAP showed the positive reaction by Western blotting method and MAP-2 was the negative reaction as an identification test of the cultured astroglia cell. It is the figure of the microscope picture which showed the state of the astroglia cell influenced by hydrogen peroxide. FIG. 4 is a photomicrograph showing that astroglia cells pretreated with guaiacol, 2M4MP and 2M4EP at each concentration (100 μM and 1 mM) are not affected when exposed to hydrogen peroxide at a concentration of 100 μM. It is the graph which showed the cell death inhibitory effect (astroglial cell survival rate) of the guaiacol under the condition which induces cell death by hydrogen peroxide. It is the graph which showed the cell death inhibitory effect (astroglial cell survival rate) of 2M4MP under the condition which induces cell death by hydrogen peroxide. It is the graph which showed the cell death inhibitory effect (astroglial cell viability) of 2M4EP under the condition which induces cell death by hydrogen peroxide.

Claims (3)

A compound that suppresses cell death of astroglial cells,
A cell death inhibitor for astroglial cells comprising a compound represented by the following general formula (I) or a salt thereof:
[In the general formula (I), R ¹ is a methyl group or an ethyl group, R ² is a hydrogen atom or a linear or side chain alkyl group having 1 to 5 carbon atoms, and R ¹ and R ² are the same. Or different. ]   2. The compound according to claim 1, wherein the compound is at least one selected from the group consisting of 2-methoxyphenol, 2-methoxy-4-methylphenol, and 2-methoxy-4-ethylphenol. Cell death inhibitor.   The cell death inhibitor according to claim 1 or 2, which is used for an anti-dementia drug, a Parkinson's disease therapeutic drug, a cerebrovascular disorder therapeutic drug, or a brain function improving drug.