JPH11246406A - Bone resorption inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject inhibitor capable of restraining osteoclast from absorbing a bone component and useful for treatment and prophylaxis of bone loss diseases e.g. osteoporosis by including a specific indole derivative as an active ingredient. SOLUTION: This inhibitor comprises a compound of formula I [R<1> and R<3> are each a hydrocarbon; R<2> is H or a (substituted) hydrocarbon; and (n) is 0 to 4], pref., a compound of formula II as an active ingredient. The inhibitor as a drum may be conventionally formed into preparations in the form of e.g. an encapsulated agent, powder agent, granular agent or pill and these preparations may be administrated orally or non-orally. Generally it is preferable to administer a dose of about 1.0 to 1,000 mg to an adult a day by dividing the dose into one to several portions. The objective inhibitor comprising a compound of formula II as an active ingredient is esp. small in cytotoxicity and useful as a prophylactic or therapeutic agent for osteoporosis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a bone resorption inhibitor, and more particularly, to a bone resorption inhibitor containing an indole derivative as an active ingredient. The bone resorption inhibitor of the present invention suppresses bone resorption by osteoclasts,
It is useful for treating or preventing osteopenia such as osteoporosis.

[0002]

2. Description of the Related Art Osteoporosis is a disease in which bone is easily broken due to a decrease in the amount of bone. Bone mass is maintained by a series of processes: bone formation by osteoblasts and bone resorption by osteoclasts, but bone formation and bone resorption are usually strictly balanced so that they are quantitatively balanced. Being controlled. However, osteoporosis occurs when bone resorption exceeds bone formation due to endocrine abnormalities and nutritional disorders.

[0003] Conventionally, estrogen agents, calcitonin, vitamin D, calcium agents and the like have been administered as therapeutic agents for osteoporosis. Further, as a compound that suppresses bone resorption, ipriflavone and a pyridine derivative described in JP-A-1-203325 are known. However, in the case of the above-mentioned therapeutic agents, due to toxicity,
It has been pointed out that the administration target is restricted or the therapeutic effect is insufficient.

[0004]

Therefore, the present invention is capable of suppressing bone resorption by osteoclasts, has low toxicity, and does not limit the dose and administration target. An object of the present invention is to provide a bone resorption inhibitor that can be used for treating or preventing osteopenia.

[0005]

Means for Solving the Problems Accordingly, the present inventors have:
In view of the development status of conventional bone resorption inhibitors, as a result of intensive research aimed at developing a drug that suppresses bone resorption, the specific indole derivative has an excellent bone resorption inhibitory action,
Further, they have found that the toxicity is extremely low, and completed the present invention based on these findings. That is, the present invention provides the following general formula (I)

[0006]

Embedded image (In the above general formula (I), R ¹ is a hydrocarbon group, R ² is a hydrogen atom or an optionally substituted hydrocarbon group, R ³ is a hydrocarbon group, and each hydrocarbon group is May be the same or different from each other, and n is 0
-4. The present invention relates to a bone resorption inhibitor containing a compound represented by the formula (1) as an active ingredient.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The active ingredient of the bone resorption inhibitor of the present invention is a specific indole derivative or a salt thereof, and can be represented by the general formula (I). In the general formula (I), R
¹ is a hydrocarbon group substituted by an aromatic ring, and may be substituted by two or more. R ² is a hydrogen atom or an optionally substituted hydrocarbon group, and R ³ is a hydrocarbon group. Each hydrocarbon group may be the same or different from each other. Further, n is an integer of 0 to 4. Therefore, the aromatic ring may not be substituted with a hydrocarbon group, or may be substituted with up to four identical or different hydrocarbon groups. As these hydrocarbon groups, those having 1 to 1 carbon atoms
20 alkyl groups; alkenyl groups having 2 to 20 carbon atoms; and aryl groups having 6 to 20 carbon atoms. Further, the aryl group may be substituted with an alkyl group. Among such hydrocarbon groups, lower alkyl groups having about 1 to 5 carbon atoms, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl and the like, especially R ³ Is preferably a methyl group.

As the compound represented by the above general formula (I), those in which R ² is a hydrogen atom and R ³ is a methyl group are particularly preferred from the viewpoint of the activity of inhibiting bone resorption. Therefore, as an active ingredient of a bone resorption inhibitor, the following formula

[0009]

Embedded image The compound represented by is suitable. The compound represented by the above general formula (I) can be produced by the method described in Synthesis, (4), 300, (1988) or a method analogous thereto, or a method of biosynthesis using a microorganism. it can.

[0010] Drugs using the bone resorption inhibitor of the present invention can be prepared in the usual manner by capsules, powders, granules, pills, tablets, suspensions, emulsions, solutions, ointments, injections, syrups or suppositories. And can be administered orally or parenterally. The administration method, dosage and number of administrations may be appropriately adjusted according to the patient's age and symptoms. Usually, about 1 to 1000 mg per day is divided into one to several doses for an adult. Good.

[Examples] Test methods showing the pharmacological effects of the compounds represented by the above formulas (hereinafter, referred to as compounds as necessary) and the results thereof will be described below.

Bone resorption inhibitory action In a collagen gel-coated culture dish, mouse-derived bone marrow cells are 4 × 10 ⁷ cells / dish, osteoblasts are 1 × 1
The 0 ⁶ cells / dish were co-cultured in a-MEM medium 15 ml. The day when the co-culture was started was defined as day 0 of culture, and the medium was replaced on days 3 and 5; at the same time, activated vitamin D was added as an agent for inducing osteoclast differentiation.
On the day, mature osteoclasts were obtained. After culturing, the enzyme inhibitor was washed away with PBS (−), and then an enzyme solution for collagen gel digestion was added thereto.
The collagen gel was digested by shaking at 0 min ^-1 . Thereafter, the mature osteoclasts were collected by centrifugation.

An ivory slice having a thickness of 0.2 mm and a diameter of 4 mm and an a-MEM containing the above compound as a sample compound
100 μl of medium was added to a 96-well plastic plate. The sample concentration was adjusted to a final concentration of 10 μg / ml. The obtained osteoclasts were appropriately diluted with an a-MEM medium, and the cell mass was well loosened. This osteoclast suspension is carefully added at 100 μl / well so that the cells are on the ivory section. After standing for 24 hours, the bone resorption fossa on the ivory section was placed in a hematoxylin solution (Sigma Chemical, St. Louis,
MO) and the number of bone resorption pits was counted under a microscope. The number of bone resorption fossa of the control containing no sample compound was P
c, The number of bone resorption pits when the sample compound was added was defined as Ps, and the rate of bone resorption inhibition was determined as the ratio of the number of bone resorption pits to the control. Bone resorption inhibition rate (%) = (Pc / Ps) × 100 Table 1 shows the bone resorption inhibition rate at a drug concentration of 10 μg / ml.

[0014]

[Table 1]

Osteoclasts derived from cytotoxic mice were dispensed into 96-well flat-bottom microplates at 5 × 10 ⁴ cells / well in 100 μl of a-MEM medium. Add the drug to be assayed to the cells and add 5% C
The cells were cultured at 37 ° C. for 2 hours in the presence of O ₂ . After confirming the cell morphology with an optical microscope, MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-d) diluted to 5 mg / ml with PBS (-) was used.
(iphenyl tetrazolium bromide) was added thereto, and the cells were further cultured at 37 ° C. for 2 hours in the presence of 5% CO ₂ . 0.04
After adding 100 μl of N hydrochloric acid-isopanol solution to dissolve the crystals, the absorbance at 595 nm was measured with a microplate reader. The absorbance of the medium alone was Ab, the absorbance without the drug treatment was Ac, and the absorbance without the drug treatment was As. Cell viability (%) = ((As-Ab) / (Ac-Ab)) × 100

The compound, which is an active ingredient of the bone resorption inhibitor of the present invention, showed a cell viability of 100% at a concentration of 10 μl / ml, and 96% at a concentration of 80 μl / ml, showing no cytotoxicity. 10μ for ipriflavone
At a concentration of 1 / ml, cell viability was 78%, 30 μl
At a concentration of / ml, remarkable cytotoxicity of 22% was observed.

Formulation Examples Formulation examples are shown below, but the present invention is not limited to these formulation examples.

Formulation Example 1 The following compound, lactose, corn starch and hydroxycellulose are mixed, kneaded by adding water, heated and vacuum dried, and then pulverized. The obtained granules are mixed with magnesium stearate and talc. Thus, a tablet of 200 mg per tablet was prepared from the following components. Composition in one tablet 50 mg Lactose 110 mg Corn starch 25 mg Hydroxycellulose 12 mg Magnesium stearate 1.5 mg Talc 1.5 mg Total 200 mg

Formulation Example 2 Tablets were prepared in the same manner as in Formulation Example 1 using the following ingredients in a conventional manner. Composition in 1 tablet 25mg Lactose 70mg Corn starch 15mg Hydroxycellulose 8mg Magnesium stearate 1mg Talc 1mg Total 120mg

Formulation Example 3 The compound, lactose, corn starch and hydroxypropylcellulose are mixed to have the following composition, kneaded by adding water, heated, vacuum-dried, and pulverized. A gelatin capsule was filled to prepare a capsule. The composition is as follows. Composition in one capsule 30mg Lactose 70mg Corn starch 45mg Hydroxypropylcellulose 5mg Total 150mg

Formulation Example 4 Sodium salicylate, sodium chloride, sodium metasulfite, methyl-paraben and propyl-paraben were mixed, distilled water was added, and the compound was dissolved to prepare an injection having the following composition. Compound 5mg Sodium salicylate 50mg Sodium chloride 180mg Sodium metasulfite 20mg Methyl-paraben 36mg Propyl-paraben 4mg Distilled water for injection 2.0ml Total 295mg

[0022]

From the above results, it can be seen that the compound which is the active ingredient of the bone resorption inhibitor of the present invention has the same action as the conventionally known bone resorption inhibitor, but has the cytotoxicity of the conventional bone resorption inhibitor. Since it is particularly less, it is understood that it is useful as an agent for preventing and treating osteoporosis.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Kenichi Mochida 1900 Sodesoshi-cho, Shimizu-shi, Shizuoka Prefecture Within the Shimizu Research Institute, Kaiyo Biotechnology Research Institute Co., Ltd. (72) Kazuo Nagai 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa Prefecture Department of Biotechnology, Tokyo Institute of Technology

Claims (2)

[Claims] 1. A compound represented by the following general formula (I): (In the above general formula (I), R ¹ is a hydrocarbon group, R ² is a hydrogen atom or an optionally substituted hydrocarbon group, R ³ is a hydrocarbon group, and each hydrocarbon group is May be the same or different from each other, and n is 0
-4. A bone resorption inhibitor comprising a compound represented by the formula (1) as an active ingredient. 2. The bone resorption inhibitor according to claim 1, wherein in the formula (I), a compound in which R ² is a hydrogen atom, R ³ is a methyl group, and n is 0 is an active ingredient.