JPH05331061A - Apoptosis-inducing agent - Google Patents

Abstract

(57) [Summary] [Structure] An apoptosis inducer containing baicalin or baicalein as an active ingredient. [Effect] According to the apoptosis inducer of the present invention, it is possible to effectively induce apoptosis in a living body. Unlike the death of cells based on necrosis, this apoptosis is a death originally incorporated in the cells themselves, and therefore unnecessary or pathogenic cells can be removed in a natural form. Therefore, it is useful as a new type of safe anti-cancer / anti-cancer agent and a therapeutic agent for many diseases caused by various virus infections.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an apoptosis-inducing agent which can be used as a medicine such as an anti-cancer agent and an antiviral agent.

[0002]

2. Description of the Related Art In recent years, a mode of apoptosis (also referred to as "apoptosis"; suicide bombing or cell necrosis) has been found and has attracted attention as to death of cell tissues.
Unlike necrosis, which is a pathological cell death, this apoptosis is considered to be a death that is originally integrated into the gene of the cell itself.

That is, a gene that programs apoptosis is activated by some external or internal factor, the programmed death protein is biosynthesized based on this gene, and the cell itself is decomposed by the generated programmed death protein. And is believed to be fatal.

[0004]

[Problems to be Solved by the Invention] If such apoptosis can be expressed in a desired tissue or cell,
Unnecessary or pathogenic cells can be eliminated naturally from the living body, which is extremely significant. However, only glucocorticoids have been known to date as compounds that induce apoptosis, and there has been a demand for the development of compounds having a superior apoptosis-inducing action.

[0005]

[Means for Solving the Problems] The inventors of the present invention have searched for various crude drug components in order to find a compound having an apoptosis-inducing action, and found that baicalin and baicalein contained in yellow stalks are excellent in inducing apoptosis. The present invention has been completed by finding that it has an action.

That is, the present invention provides an apoptosis inducer containing baicalin or baicalein as an active ingredient.

In the present invention, baicalin and baicalein used as the active ingredients are represented by the following formula in which R is GlcA.
(Baicalin) and R are compounds represented by H (Baicalein).

[0008]

[Chemical 1]

These baicalin and baicalein are
It is a known compound and is known to have anti-inflammatory action, anti-allergic action, and antihypertensive action. Also,
It is also known to inhibit the action of reverse transcriptase (Japanese Patent Laid-Open No. 1-163120), but it is completely known that these compounds induce apoptosis as in the present invention and eliminate unnecessary or pathogenic cells. Absent.

The agent for inducing apoptosis of the present invention is prepared by combining the above baicalin or baicalein with a known pharmaceutical carrier as it is to prepare an oral preparation such as tablets, powders, granules and liquids, injections and infusions. It can be prepared by using the above-mentioned parenteral preparations, and further suppositories.

The pharmaceutical carrier can be selected according to the above-mentioned administration form and dosage form. In the case of oral preparations, for example, starch, lactose, sucrose, mannitol, carboxymethyl cellulose, corn starch, inorganic salts and the like are used. It When preparing an oral preparation, a binder, a disintegrant,
Surfactants, lubricants, fluidity promoters, flavoring agents, coloring agents, fragrances and the like can be added. Specific examples of these include the following.

(Binder) Starch, dextrin,
Gum arabic powder, gelatin, hydroxypropyl starch, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, crystalline cellulose, ethyl cellulose, polyvinylpyrrolidone, macrogol.

(Disintegrating agent) Starch, hydroxypropyl starch, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, carboxymethyl cellulose, low-substituted hydroxypropyl cellulose.

(Surfactant) Sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polysorbate 80.

(Lubricant) Talc, wax, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol.

(Flowability Accelerator) Light anhydrous silicic acid, dried aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate.

Further, liquid preparations for oral use may be suspensions, emulsions, syrups and elixirs, and these various dosage forms may be mixed with a flavoring agent, a flavoring agent and a coloring agent.

On the other hand, in the case of parenteral preparations, distilled water for injection, physiological saline, glucose solution, vegetable oil for injection, sesame oil, peanut oil, which is the active ingredient of the present invention, is used as a diluent in the conventional manner. It is prepared by dissolving or suspending it in soybean oil, corn oil, propylene glycol, polyethylene glycol, etc., and adding a bactericide, preservative, stabilizer, tonicity agent, soothing agent, etc., if necessary. ..

The dose of this apoptosis-inducing agent varies depending on the route of administration, the degree of disease, the age of the recipient, and the like. Generally, in the case of oral administration, it is 100 m per adult per day.
An amount of about g to 6 g may be administered in 1 to 3 divided doses.
In the case of a parenteral preparation, about 1 to 100 mg may be administered daily.

It should be noted that baicalin and baicalein used in the present invention are extremely safe, as is apparent from the fact that no death occurs in oral administration of 1 g / kg, which is the limit of administration, to mice and rats. It is one that can be used with confidence.

[0021]

EXAMPLES The present invention will be described in more detail with reference to test examples and examples, but the present invention is not limited to these examples.

Test Example 1 Examination of Cellular DNA Fragmentation Action on Virus-Infected Cells: (Test Method) The DNA fragmentation action of baicalin and baicalein was examined as follows. Falcon 3084 flask (75 cm ² ) with 10% FCS-
20 ml of virus-infected cells, MT4, which was adjusted to 1 × 10 ⁶ cells / ml with RPMI1640 medium, was placed in 4 m.
DMSO solutions containing test reagents at concentrations of g / ml, 400 μg / ml and 40 μg / ml were added in 100 μl portions.

Culturing was carried out at 37 ° C. in a carbon dioxide incubator,
After 1 hour, collect 10 ml and transfer to a centrifuge tube.
It was centrifuged at 000 rpm for 6 minutes. After centrifugation, the supernatant was discarded and the precipitated cells were collected. The remaining 10 ml was further cultivated for 2 hours, and the cells were collected by the same centrifugation as above (culture time 3 hours). Each of the precipitated cells was immediately frozen and stored at -80 ° C until DNA extraction.

Extraction of DNA is carried out by isoquick (IsoQ
uick ^™ ; vendor Tanehashi) was used as follows. That is, the precipitated cells that had been frozen and stored were
After suspending in μl Regent-A, 100 μl Regent-1 (Lysis Solution) was added to lyse the cells. Add 700 μl of Regent-
2 (Extraction Matrix) and 400 μl
Regent-3 (Extraction buffer)
Was added and the mixture was vortexed.

After centrifuging this mixed solution at 15,000 rpm for 5 minutes, the aqueous phase was recovered and 1/4 amount of Regent-4 was collected.
(Sodium acetate solution) was added, the same volume of isopropyl alcohol was further added thereto, and the mixture was lightly stirred to precipitate DNA. 15,000 r of mixed solution with DNA deposited
After centrifugation at pm for 10 minutes and removing the supernatant, DN of the sediment
A was further centrifugally washed with 70% ethanol. Obtained D
After NA was dried up, it was dissolved in 20 μl of TE buffer and subjected to agarose gel electrophoresis. Electrophoresis was carried out using a Nu Live agarose 3% gel for 10
It was carried out at 0 V for 50 minutes. The marker is ф × 174 / H
The aeIII digest was used.

(Result) Figure (photograph) of the result of electrophoresis
Shown in. In the figure, codes 7 to 12 on the upper side show DNA fragmentation when baicalein is allowed to act (code 7:
0.2 μg / ml, 1 hour action, code 8: 2 μg / m
1, 1 hour action, Code 9: 20 μg / ml, 1 hour action, Code 10: 0.2 μg / ml, 3 hour action, Code 11: 2 μg / ml, 3 hour action, Code 12: 2
0 μg / ml for 3 hours).

In the figure, the lower cords 13 to 18 are
DNA fragmentation when treated with baicalin (code 13: 0.2 μg / ml, action for 1 hour, code 14: 2
μg / ml, 1 hour action, Code 15: 20 μg / m
l, 1 hour action, Code 16: 0.2 μg / ml, 3 hour action, Code 17: 2 μg / ml, 3 hour action, Code 18: 20 μg / ml, 3 hour action), Codes 19 and 20 are control DNA The result of fragmentation is shown.

As is clear from these results, DNA of MT4 (HT cell-derived HTLV-infected cell line), which is a virus-infected cell, is fragmented by baicalein and baicalin at an integral multiple of 200 bp, and apoptosis is induced. It was revealed what was done. In particular, baicalin induced apoptosis even at low concentrations of 0.2 μl /.

Example 1 Preparation of Condyle Granules: 10 g of baicalin was mixed with 89 g of lactose and 1 g of magnesium stearate, and the mixture was tabletted with a single-shot tableting machine to give a diameter of 20 mm and a weight of about 2.3 g. I made a slug tablet. The slug tablet was crushed with an oscillator, sized and sieved to obtain a granule having a particle size of 20 to 50 mesh. (1g of this granule contains 100mg of baicalin, and
Take 2.5 g in several divided doses. )

Example 2 Preparation of tablet preparation: 2.5 g of baicalin was mixed with 20 g of microcrystalline cellulose and 5 g of magnesium stearate, and the mixture was tableted with a single-shot tableting machine to give a diameter of 7 m.
m, weight 225 mg tablets were produced. One tablet of this tablet contains 20 mg of baicalin. (This tablet
Adults take 5-7 tablets in several divided doses per day. )

Example 3 Preparation of capsule agent: 10 g of baicalein was uniformly mixed with 89.5 g of cornstarch and 0.5 g of light anhydrous silicic acid, and 200 mg thereof was filled in a No. 2 capsule,
A capsule was prepared. (One capsule of this capsule contains 20 mg of baicalein, and 5 to 7 capsules are taken in divided doses per day for an adult.)

Example 4 Preparation of injection: 1 part glucose in 1 part distilled water for injection
00 mg and 10 mg baicalin were dissolved, and the total amount was adjusted to 15 ml with distilled water for injection, then, the mixture was poured into an ampoule of 5 ml, and heat sterilized at 121 ° C. for 15 minutes to obtain an injection.

[0033]

INDUSTRIAL APPLICABILITY The apoptosis-inducing agent of the present invention containing baicalin or baicalein as an active ingredient can effectively induce apoptosis in a living body. Unlike the death of cells based on necrosis, this apoptosis is a death originally incorporated in the cells themselves, so that unnecessary or disease-causing cells can be naturally removed.

Conventionally, for example, carcinostatic agents and anticancer agents for cancer include alkylating agents, antimetabolites, antibiotics, and DNA.
Drugs such as synthetic inhibitors and immune enhancers are used,
Of these, alkylating agents, antimetabolites, antibiotics, D
Drugs such as NA synthesis inhibitors have a problem that they affect not only cancer cells but also normal cells, and that side effects are too strong, and immunopotentiators have a weak effect.

However, the use of the apoptosis inducer of the present invention can induce programmed apoptosis in cancer cells and kill the immortalized cancer cells. It can be advantageously used as an anti-cancer agent. Further, since it can induce apoptosis in tissues and cells infected with various viruses and remove these tissues and cells, it is useful for treating many diseases caused by viral infection.

[Brief description of drawings]

FIG. 1 is a photograph showing DNA of MT4 cell line fragmented by the addition of baicalein and baicalin.

[Procedure amendment]

[Submission date] July 3, 1992

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] 0002

[Correction method] Change

[Correction content]

[0002]

2. Description of the Related Art In recent years, a mode of apoptosis (also called apoptosis or apoptoses; self- destruction or cell self-destruction ) has been found and is drawing attention as to death of cell tissues. Unlike necrosis, which is a pathological cell death, this apoptosis is considered to be a death that is originally integrated into the gene of the cell itself. ─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] March 1, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Brief explanation of the drawing

[Correction method] Change

[Correction content]

[Brief description of drawings]

FIG. 1 is an electrophoretic photograph showing DNA of MT4 cell line fragmented by addition of baicalein and baicalin.

Claims (1)

[Claims] 1. An apoptosis inducer containing baicalin or baicalein as an active ingredient.