JP2005126371A - Agent for osteometabolic disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a compound enabling osteometabolic diseases including osteoporosis to be prevented or treated by maximally stopping the perishment of osteoblast by hydrogen peroxide to maintain the absolute number of the osteoblast at high levels to yield osteoplastic predominance. <P>SOLUTION: The agent for osteometabolic diseases comprises a compound of the general formula(I)( wherein, R<SP>1</SP>and R<SP>2</SP>are each a 1-5C straight-chain or side-chain alkyl ) or a salt thereof. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an agent for bone metabolic disease, and in particular, bone metabolic disease used for the prevention or treatment of bone metabolic diseases (bone metabolic disorders) such as osteoporosis, Behcet's disease, osteomaltosis and diabetic osteopenia. It relates to the preparation.

  Osteoporosis, one of the bone metabolic diseases, is said to have about 5 to 8 million osteoporosis patients in Japan and is increasing year by year. As is well known, this disease is a disease in which calcium deficiency progresses, bone density decreases, and the bone becomes scary and easily breaks. That is, there are two types of bone: osteoclasts that dissolve bone calcium to destroy old bones, and osteoblasts that repair and regenerate broken bones. When the balance between the two is lost and bone lysis (bone resorption) by osteoclasts exceeds bone regeneration by osteoblasts, bone calcium deficiency progresses, bone density decreases, and bone metabolism such as osteoporosis Sexual illness is caused.

In contrast, activated vitamin D3 and calcitriol that increase calcium absorption from the intestinal tract and promote bone formation, calcitonin and elcatonin that inhibit bone resorption and promote bone formation, or inhibit bone resorption and estrogen calcitonin secretion A therapeutic agent such as ipriflavone that promotes has been used, and some results have been obtained (for example, Patent Document 1). However, the situation is serious because the number of domestic patients with osteoporosis is very high, 5 million to 8 million people, and this disease can cause patients to sleep, especially the elderly, and eventually dementia. Therefore, a more effective preventive means or therapeutic means has been desired.
Japanese Patent Laid-Open No. 7-17854

By the way, it is said that 50 to 70% of osteoblasts die during the process of bone formation and the number thereof decreases. One of the causes is said to be hydrogen peroxide (H ₂ O ₂ ) produced in the body. That is, it is reported that when osteoblasts are exposed to hydrogen peroxide produced in the body, the osteoblasts are induced in the direction of cell death. In view of reestablishing the balance lost in bone metabolism (or preventing the balance from being lost), the present inventor has focused on the death of osteoblasts. In other words, it is effective to prevent or treat bone metabolic diseases by minimizing the death of osteoblasts caused by hydrogen peroxide and maintaining its absolute number at a high level, thereby providing an osteogenic advantage. Various compounds with such an action (which can avoid (prevent) osteoblast 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 osteoblasts, and arrived at the present invention.

［ただし、上記一般式におけるＲ^１およびＲ^２は、いずれも炭素数１〜５の直鎖または側鎖のアルキル基であって、Ｒ^１とＲ^２は同一であっても異なっていてもよい］。 The bone metabolic disease agent according to claim 1 is characterized by containing a compound represented by the following general formula (I) or a salt thereof.

[However, R ¹ and R ² in the above general formula are both straight-chain or side-chain alkyl groups having 1 to 5 carbon atoms, and R ¹ and R ² may be the same or different. ].

  According to the present invention, it is possible to minimize the death of osteoblasts (decrease in the number of osteoblasts), which is said to be 50 to 70% lost during the bone formation process. As a result, it is possible to prevent or treat bone metabolic diseases. When used in combination with another drug having another mechanism (for example, a drug that suppresses the activity of osteoclasts), bone formation becomes more dominant, and bone metabolic diseases can be effectively prevented or treated.

As described above, the bone metabolic disease agent of the present invention contains a compound represented by the following general formula (I) or a salt thereof.

R ¹ and R ² in the general formula (I) are both linear or side chain alkyl groups having 1 to 5 carbon atoms, and R ¹ and R ² may be the same or different. . In addition, it is preferable that R ¹ has 1 to 2 carbon atoms from the viewpoint of excellent ability to prevent the death of osteoblasts, and more preferably a methyl group. R ² preferably has 1 to 3 carbon atoms from the viewpoint of excellent ability to prevent the death of osteoblasts, and more preferably a methyl group or an ethyl group. R2 is preferably bonded to the 3 or 4 position from the viewpoint of excellent ability to prevent the death of osteoblasts, and more preferably bonded to the 4 position. Specific examples include 2-methoxy-3-methylphenol, 2-methoxy-3-ethylphenol, 2-methoxy-3-propyl (or isopropyl) phenol, 2-methoxy-3-butyl (or isobutyl) phenol, 2-methoxy-4-methylphenol, 2-methoxy-4-ethylphenol, 2-methoxy-4-propyl (or isopropyl) phenol, 2-methoxy-4-butyl (or isobutyl) phenol, 2-methoxy-5 Methylphenol, 2-methoxy-5-ethylphenol, 2-methoxy-5-propyl (or isopropyl) phenol, 2-methoxy-5-butyl (or isobutyl) phenol, 2-ethoxy-3-methylphenol, 2-ethoxy -3-ethylphenol, 2-ethoxy 3-propyl (or isopropyl) phenol, 2-ethoxy-3-butyl (or isobutyl) phenol, 2-ethoxy-4-methylphenol, 2-ethoxy-4-ethylphenol, 2-ethoxy-4-propyl (or isopropyl) ) Phenol, 2-ethoxy-4-butyl (or isobutyl) phenol, 2-ethoxy-5-methylphenol, 2-ethoxy-5-ethylphenol, 2-ethoxy-5-propyl (or isopropyl) phenol, 2-ethoxy -5-butyl (or isobutyl) phenol and the like. Among them, 2-methoxy-4-methylphenol (hereinafter, also simply referred to as “2M4MP”) in that it has an excellent ability to prevent the death of osteoblasts. 2-methoxy-4-ethylphenol (hereinafter, simply "2M4EP"), 2-ethoxy-4-methylphenol (hereinafter also simply referred to as "2E4MP") and 2-ethoxy-4-ethylphenol (hereinafter also simply referred to as "2E4EP") are preferred, among which 2M4MP Most preferred are 2M4EP and 2E4MP. 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 2M4MP and 2M4EP are both constituents 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, creosote is used for intestinal preservatives and has an expectorant effect by inhalation indication. In 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 creosote is used as a disinfectant for external use and an expectorant for internal use. Yes.

Salt Form The agent for bone metabolic disease of the present invention can be in the form of a salt 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, amines (monoethanolamine) , Diethanolamine, triethanolamine), ammonium salts and the like.

Bone metabolic disease Examples of the target disease name using the agent for bone metabolic disease of the present invention include osteoporosis, hypercalcemia, Behcet's disease, osteomaltosis, diabetic osteopenia and the like.

Amount used The dose (use amount) of the agent for bone metabolic disease of the present invention varies depending on the type or sex of the target animal, sex, age, and symptom level. In the case of intrarectal 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, and in the case of administration as an injection. Is 0.05 to 5 mg, preferably 0.25 to 2.5 mg, per 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 agent for bone metabolic disease 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 agent for bone metabolic disease of the present invention can be taken not only as a pharmaceutical product but also as a general food and 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), paste 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.) Ingestion of the agent for bone metabolic disease of the present invention can also be achieved.

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

Osteoblast culture (see FIGS. 1 and 2)
Osteoblasts were cultured by a conventionally known method. That is, a skull extracted from a newborn rat (1-2 days of age) was placed in a Hanks solution containing 0.25% trypsin solution and 0.1% collagenase solution (10 minutes at 37 ° C. for 5 times). ). Subsequently, it culture | cultivated by the alpha-MEM culture solution containing 10% fetal calf serum (FCS), and adjusted to 1 * 10 < ⁴ > piece / cm < ² >. The next day, the cells were cultured in an α-MEM culture medium containing 10% FCS, ascorbic acid and β-glycerophosphate. The culture medium was changed every other day, and the number of osteoblasts was measured after 28 days (in 1 well). Further, in order to confirm the growth of osteoblasts, the calcium concentration during the 28 days was measured (in 1 well) (see FIG. 3). As shown in FIG. 3, since the amount of calcium (in 1 well) increases with time, alkaline phosphatase activity as an index of osteoblast differentiation also increases with time. It can be seen that osteoblasts are grown by this culture method.

Influence of osteoblasts by hydrogen peroxide Exposure to osteoblasts with varying concentrations of hydrogen peroxide was performed, and cell viability after 6 hours was measured (in 1 well). As a result, as shown in FIG. 4, cell death was noticeable from about 300 μM, and significant cell death was confirmed at 500 μM and 1000 μM. Accordingly, as a future experiment, the concentration of hydrogen peroxide that causes cell death was set to 500 μM.

Examples 1-3 and Comparative Examples 1-5
FIG. 5 shows the basis for setting the amount of 2M4MP, 2M4EP, etc. used in the following examples to 1 mM. That is, in the situation where cell death is induced by hydrogen peroxide, when the usage amount of 2M4MP, 2M4EP, etc. is 1 mM, the cell death inhibitory effect (osteoblast survival rate) is at a high level as shown in FIG. Admitted.

Example 1 (Induction of cell death by hydrogen peroxide and inhibition of cell death by 2M4MP)
FIG. 6 (a) is a schematic explanatory diagram showing the growth state of untreated osteoblasts (after 7 days) as viewed with a microscope. (B) is a schematic explanatory drawing showing the growth state after 6 hours of exposure to hydrogen peroxide (500 μM) (discolored black when osteoblasts die). (C) is a schematic explanatory drawing showing the growth state after 6 hours of exposure to 2-methoxy-4-methylphenol (1 mM), and (d) shows hydrogen peroxide 500 μM and 2M4MP (Tokyo Chemical Industry ( It is a schematic explanatory drawing which shows the growth state 6 hours after exposure (simultaneous administration) to which 1 mM (made by Tokyo Chemical Industry first grade 2-methoxy-4-methylphenol) was exposed.

  As is apparent from FIGS. 6A to 6D, osteoblast cell death is caused by hydrogen peroxide (b). However, in the system (d) in which hydrogen peroxide and 2M4MP are used in combination, since osteoblasts continue to live, it is considered that 2M4MP blocked the induction of hydrogen peroxide cell death. Shows that it has the effect of preventing cell death. In addition, since the osteoblast cell death is not induced in the system (c) of 2M4MP alone, the toxicity of 2M4MP is low and there is no problem.

Example 2 (Induction of cell death by hydrogen peroxide and inhibition of cell death by 2M4EP)
FIG. 7 (a) is a schematic explanatory diagram showing a growth state of untreated osteoblasts (after 7 days) as viewed with a microscope. (B) is a schematic explanatory drawing showing the growth state after 6 hours of exposure to hydrogen peroxide (500 μM) (discolored black when osteoblasts die). (C) is a schematic explanatory view showing the growth state after 6 hours of exposure to 2-methoxy-4-ethylphenol (1 mM), and (d) shows 500 μM hydrogen peroxide and 2M4EP (Tokyo Chemical Industry ( It is explanatory drawing which shows the growth state 6 hours after exposed to 1 mM of (Co., Ltd. product, Tokyo Chemical Industry first grade 2-methoxy-4-ethylphenol).

  As is clear from FIGS. 7A to 7D, osteoblast cell death is caused by hydrogen peroxide (b). However, in the system (d) in which hydrogen peroxide and 2M4EP are used in combination, since osteoblasts continue to live, it is considered that 2M4EP blocked the induction of hydrogen peroxide cell death. Shows that it has the effect of preventing cell death. In addition, since the osteoblast death was not induced in the system (c) of 2M4EP alone, the toxicity of 2M4EP is low and there is no problem.

Comparative Example 1 (Action of Pseudo Skeletal Compound Guayacol)
FIG. 8 (a) is a schematic explanatory diagram showing a growth state of untreated osteoblasts (after 7 days) as viewed with a microscope. (B) is a schematic explanatory drawing showing the growth state after 6 hours of exposure to hydrogen peroxide (500 μM) (discolored black when osteoblasts die). (C) is a schematic explanatory diagram showing the growth state after 6 hours of exposure to guaiacol (2-methoxyphenol) (1 mM), and (d) is a graph showing hydrogen peroxide (500 μM) and guaiacol (1 mM). It is a schematic explanatory drawing which shows the growth state 6 hours after exposure.

  As is apparent from FIGS. 8A to 8D, osteoblast cell death is caused by hydrogen peroxide (b), and osteoblast cell death due to the hydrogen peroxide can be prevented by guaiacol. It was not possible (d).

Comparative Examples 2 to 5 (action of a pseudo skeleton compound other than guaiacol) and Example 3 (cell death inhibitory action of another compound of the present invention (2E4MP))
The guaiacol used in Comparative Example 1 was o-cresol (2-methylphenol) as Comparative Example 2, p-cresol (4-methylphenol) as Comparative Example 3, phenol as Comparative Example 4, and thymol (Comparative Example 5). Except for changing to 2-isopropyl-5-methylphenol), the same operation as in Comparative Example 1 was performed, and the inhibitory action (inhibitory action) on the induction of cell death by hydrogen peroxide was examined. The results are shown in FIG. 9 together with the results of other compounds (2-ethoxy-4-methylphenol (2E4MP, used at 1 mM and 100 μM)) included in the general formula (I) in the present invention.

  As is clear from FIG. 9, no inhibitory effect on cell death was observed in any of the above-mentioned compounds similar to the general formula (I) in the present invention (which is also clear compared to the system using 100 μM 2E4MP). .

From Examples 1-3 and Comparative Examples 1-5, it can be said that the effect which prevents the cell death of a bone field cell is a specific effect of the compound in this invention. The structural formulas of the compounds of the present invention used in Examples 1 to 3 and the comparative compounds (pseudo skeleton compounds) used in Comparative Examples 1 to 5 are summarized in [Table 1] below.

Formulation Example 1 (pills)
(Prescription 1)
2M4MP or 2M4EP 5mg
Daylily 25mg
Glycerin 10mg
Ordinary water 50mg
Each component of the following prescription is kneaded, the pill lump is divided with a cutting machine , and is made with a round machine , and a pill containing 5 mg of the bone metabolic disease agent of the present invention is contained in one round. Prepared.

Formulation Example 2 (Hard capsule)
(Prescription 2)
2M4MP or 2M4EP 10mg
Starch 250mg
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 bone metabolic disease agent of the present invention in one capsule.

Formulation Example 3 (Soft capsule)
(Prescription 3)
2M4MP or 2M4EP 10mg
Olive oil 200mg
A solution was obtained by dissolving in 2M4MP or 2M4EP and olive oil and filled into soft capsules to prepare soft capsules containing 10 mg of the bone metabolic disease agent of the present invention in one capsule.

Formulation Example 4 (tablet)
(Prescription 4)
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, it is possible to minimize the death of osteoblasts (decrease in the number of osteoblasts), which is said to be 50 to 70% lost during the bone formation process. As a result, it is possible to prevent or treat bone metabolic diseases. It can be used alone or in combination with other drugs with different mechanisms (for example, drugs that suppress the activity of osteoclasts). Can be prevented or treated.

It is the schematic explanatory drawing which showed the culture method of the osteoblast. It is the flowchart figure which showed the exchange time of the alpha-MEM culture solution, and the measurement time of the amount of calcium in the culture of osteoblasts for 28 days. It is the graph which showed the increase in alkaline phosphatase activity and the amount of calcium in the culture | cultivation of the osteoblast for 28 days. It is the graph which showed the condition of the cell death performed by changing the density | concentration of hydrogen peroxide. It is the graph which showed the cell death inhibitory effect (osteoblast survival rate) of 2M4MP and 2M4EP under the condition where the cell death was induced by hydrogen peroxide, which is the basis for the experimental use amount of 1 mM. FIG. (A) is a schematic explanatory diagram showing the growth state of untreated osteoblasts under a microscope, (b) is a schematic explanatory diagram showing the growth state 6 hours after exposure to hydrogen peroxide, (c) ) Is a schematic explanatory diagram showing the growth state 6 hours after exposure to 2M4MP, and (d) is a schematic explanatory diagram showing the growth state 6 hours after exposure to hydrogen peroxide and 2M4MP. (A) is a schematic explanatory diagram showing the growth state of untreated osteoblasts under a microscope, (b) is a schematic explanatory diagram showing the growth state 6 hours after exposure to hydrogen peroxide, (c) ) Is a schematic explanatory diagram showing the growth state 6 hours after exposure to 2M4EP, and (d) is a schematic explanatory diagram showing the growth state 6 hours after exposure to hydrogen peroxide and 2M4MP. (A) is a schematic explanatory diagram showing the growth state of untreated osteoblasts under a microscope, (b) is a schematic explanatory diagram showing the growth state 6 hours after exposure to hydrogen peroxide, (c) ) Is a schematic explanatory view showing the growth state after 6 hours of exposure to guaiacol, and (d) is a schematic explanatory view showing the growth state after 6 hours of exposure to hydrogen peroxide and guaiacol. It is the graph which showed the cell death inhibitory effect (osteoblast survival rate) of o-cresol, p-cresol, a phenol, thymol, and 2E4MP (1 mM, 100 micromol) in the condition which induced cell death by hydrogen peroxide. .

Claims (2)

A bone metabolic disease agent comprising a compound represented by the following general formula (I) or a salt thereof:

[However, R ¹ and R ² in the above general formula are both straight-chain or side-chain alkyl groups having 1 to 5 carbon atoms, and R ¹ and R ² may be the same or different. . ]   The compound is at least one selected from the group consisting of 2-methoxy-4-methylphenol, 2-methoxy-4-ethylphenol, and 2-ethoxy-4-methylphenol. 1. The agent for bone metabolic disease according to 1.

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