JP3525182B2 - New anti-leukemia cell agent - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel anti-leukemic cell agent containing a plant-derived polyphenol as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION Leukemia is a disease in which white blood cells in the bone marrow proliferate autonomously, a so-called blood cancer, which causes anemia, leukocytosis, thrombocytopenia, etc., often in the liver, spleen,
It is known to create secondary infiltrative lesions (metastasis) or secondary hematopoietic lesions in other organs such as lymph nodes.

As therapeutic agents for such leukemias, various synthetic substances, antibiotics, naturally-occurring substances, interferons, interleukins, TNF (tumor necrosis factor), CSF (colony formation stimulating factor) inhibitors and the like have hitherto been used. Biotechnology products are being developed. These act on leukemia cells to inhibit their growth,
The disease is treated by causing necrosis or necrosis. However, the cell death due to the necrosis often has a serious defect that not only the diseased cells but also the surrounding normal cells cause a new disease. Therefore, without affecting the surrounding normal cells,
A therapeutic agent that acts only on pathological cells and inhibits the proliferation or necrosis thereof is desirable.

Recently, an apoptosis-inducing anti-leukemic cell agent has been found as a therapeutic agent satisfying such demands,
Until now, Acrocybe cyl
Lectin protein extracted from the pulp of Indracea, Azuki bean (Abrus precatori)
(us) lectin protein isolated from seeds, hinokitiol extracted from cypress family plants, and the like have been proposed. Here, apoptosis is a life phenomenon in which, when it is disadvantageous for cells that have been damaged to be difficult to repair to survive, the cells actively self-digest according to a series of programs. When an apoptosis-inducing anti-leukemia cell is used, leukemia cells in blood selectively autolyze, so that leukemia can be treated in a state in which there is little influence on normal cells.

On the other hand, recently, polyphenols contained in tea, soybeans, persimmons, grapes, etc. have been found to be effective in eliminating active enzymes causing various diseases and in controlling other ecological conditions. Development of various applications utilizing its physiological activity is under way. This polyphenol is a general term for compounds having a plurality of phenolic hydroxyl groups in the molecule, and is present as a plant component and as a metabolite in almost all land plants. The main component is a flavonoid, that is, a basic structure of two benzene rings (A ring and B ring) bonded via a pyran ring (C ring). Further, depending on the bonding state of these A ring, C ring and B ring, the presence or absence of double bond, the number of hydroxyl groups and the bonding position thereof, flavone, flavonol, flavanone, isoflavone,
It is classified into catechin, chalcone, and anthocyanin. Of these, the ones that have received physiological and pharmacological attention are the formulas

[Chemical 1] A catechin and its derivative having a flavan-3-ol structure represented by: This molecule has a property of polymerizing, and two or more molecules are bound to each other and are present in nature. For example, tea catechin is a monomer, but dimers and trimers are present in grape seeds and immature apple fruits as proanthocyanidins, oligomeric proanthocyanidins and the like .

[0006]

The present invention has been made for the purpose of obtaining a novel anti-leukemic cell agent, particularly a novel anti-leukemic cell agent capable of inducing apoptosis, from readily available plant-derived polyphenols. It is a thing.

[0007]

Means for Solving the Problems The present inventors have hitherto aimed to provide animal or plant-derived lectins or protease inhibitors as functional materials for medical use, or to improve cell agglutination properties against established leukemia cells. As a result of various studies on cytotoxicity, it was found that certain plant-derived polyphenols have an excellent growth inhibitory effect on leukemia cells, and this is caused by apoptosis depending on the type of polyphenols. Therefore, the present invention has been completed based on this finding.

That is, the present invention is directed to green tea, red peony root or ground.
Prussanidin B-3, EGCG-2C8, and E derived from 楡
Apoptosis-inducing anti-leukemia cell agent comprising polyphenols consisting of GCG-C16est or Sangiin H-6 as an active ingredient, and cinnamon-derived procyanidin C-
The present invention provides an anti-leukemic cell agent containing 1 as an active ingredient.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The polyphenols used in the present invention are procyanines derived from green tea, red peony root, or ground peony.
Gin B-3, EGCG-2C8, EGCG-C16es
t, Sangiin H-6 and cinnamon-derived procyanidin C
-1 .

These can be easily extracted according to a method commonly used for extracting various plant components.

EGCG-2C8 and EGCG-C1 used as fat-soluble derivatives of predetermined polyphenols
6est can be easily synthesized by reacting a metal salt of a predetermined polyphenol with a predetermined long-chain alkyl halide.

Examples of polyphenols used in the present invention include those represented by the following formulas (A) to (E) . (A) Procyanidin B-3

[Chemical 2] (B) Procyanidin C-1

[Chemical 3] (C) EGCG-2C8

[Chemical 4] (D) EGCG-C16est

[Chemical 5] (E) Sanguiin H-6

[Chemical 6]

Next, the antileukemic cell activity of polyphenols was measured by measuring the amount of ATP in the cells, and the apoptosis induction activity was measured by agarose gel electrophoresis.
Confirmed by NA analysis.

[0014] In this way, up Roshianijin B-3,
EGCG-2C8, EGCG-C16est, antileukemic cell activity of Sa Ngii <br/> emissions H-6 is due to apoptosis, procyanidin C-1 is found to be those not due to apoptosis It was

Furthermore, polyphenols having antileukemic cell activity, for example, procyanidin B-3, procyanidin C-1, Sa Ngiin H-6, E GCG-2C8 ,
On the etc. EGCG-C16es t showing the significant anti-leukemic cell activity, shows the activation activity against normal cells other than E GCG-2C8.

Thus, the anti-leukemic cell agent of the present invention is
Since it has a remarkable growth inhibitory activity on leukemia cells and also has a remarkable activation activity on normal cells, it is suitable as a therapeutic agent for leukemia. When it is used as a therapeutic agent for leukemia, it may be administered orally or parenterally. The dose may vary depending on the degree of leukemia, but is usually 0.01 to 10 mg per day.
It is selected within the range of / kg. Since this compound exhibits a leukemia cell growth inhibitory action by inducing apoptosis, a sufficient effect can be obtained by administration in a smaller amount than other cell growth inhibitors. When this is administered parenterally, 0.1
Although it is formulated as an aqueous solution having a concentration of 10%, a solubilizing agent, a buffering agent, an isotonicity agent, a stabilizer, a preservative,
Additives commonly used for injection solutions such as soothing agents can be used in combination. When administered orally, it is usually formulated in the form of a liquid formulation, but if desired, it can be formulated into granules, tablets, troches, etc. by adding an appropriate excipient.

[0017]

EXAMPLES Next, the present invention will be described in more detail by way of examples.

EXAMPLE Three types of leukemia cells, Jurkat cells (derived from acute lymphocytic leukemia), ML-2 cells (derived from acute myeloid leukemia), and SupT-1 cells (derived from lymphoma), and normal cells were used. using 36-year-old man from peripheral blood white blood cells, respectively as a cell, were tested by the following methods cytotoxicity for indicates to polyphenol in Table 1.

[0019]

[Table 1]

First, each cell (2.22 × 10 ⁵ cells / m 2)
1) was cultured in RPMI-1640 medium at 37 ° C., 50 μl per well was distributed to a 96-well plate, and then 5%
It was aged at 37 ° C. for 24 hours in a humid atmosphere containing CO ₂ . Then, a predetermined polyphenol solution (10 μl per well) was injected, and in a moist atmosphere containing 5% CO ₂ ,
After culturing at 37 ℃ for 48 hours, 50μ per well
1 ATP extractant was added, and the mixture was incubated at room temperature for 10 seconds, and 50 μl of the luciferin / luciferase solution was added to the supernatant of each well, and Microlumart LB96P (EG
& G BERHOLD), ATP of each cell
The cell viability was determined by measuring the amount. The results are shown in FIGS. 1 to 5 as a graph showing the relationship between each polyphenol amount (μg) and cell viability (%). As is clear from these figures, these leukemic cytotoxicity
In polyphenols, the EGCG-2C8 survival for both Jurkat cells and normal cells 0%
Shows a strong cytotoxicity reduced to, while others positive
It exhibits activating activity on normal cells. As a result of DNA analysis of Jurkat cells treated with polyphenols by agarose gel electrophoresis, there is a cytotoxicity of each polyphenol from DNA fragmentation due to apoptosis induction and not due to apoptosis induction. confirmed.

[0021]

INDUSTRIAL APPLICABILITY According to the present invention, an anti-leukemic cell agent which is highly toxic to leukemia cells and which has an activating activity on normal cells can be obtained from easily available raw materials.

[Brief description of drawings]

FIG. 1 is a graph showing the cytotoxicity of procyanidin B-3 .

FIG. 2 is a graph showing the cytotoxicity of procyanidin C-1 .

FIG. 3 is a graph showing the cytotoxicity of EGCG-2C8 .

FIG. 4 is a graph showing the cytotoxicity of EGCG-C16est .

FIG. 5 is a graph showing the cytotoxicity of Sangiin H-6 .

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI A61P 35/02 A61P 35/02 C07D 311/62 C07D 311/62 // C07H 13/08 C07H 13/08 (72) Inventor Moriwaki Sawako 807 Nonoshita, Yadomachi, Tosu City, Saga Prefecture 1 Kyushu Institute of Industrial Technology, Institute of Industrial Science (72) Inventor Takashi Tanaka 1-14 Bunkyocho, Nagasaki City, Nagasaki Prefecture Faculty of Pharmaceutical Sciences, Nagasaki University (56) Reference JP 4 -264026 (JP, A) JP-A-7-330599 (JP, A) JP-A-2001-97872 (JP, A) JP-A-2001-199881 (JP, A) International Publication 97/036497 (WO, A1) Anticancer Res ． , 1996, Vol. 16. , No. 4A, p p. 1943-1946 Eur. J. Pharmacol, 1999, Vol. 381, No. 2-3, pp. 171-183 Biosci. Biotechno l. Biochem, 1998, Vol. 62, No. 8, pp. 1483-1487 Arch. Pharmaceutical Res. , 1995, Vol. 18, No. 6, pp. 396-401 (58) Fields surveyed (Int.Cl. ⁷ , DB name) A61K 35/78 A61K 31/353 A61K 31/7004 A61K 31/702 A61K 31/7034 BIOSIS (STN) CA (STN) MEDLINE (STN) )

Claims (2)

(57) [Claims] 1. Procyanani derived from green tea, red peony root or earthen worm
Gin B-3, EGCG-2C8, EGCG-C16es
An apoptosis-inducing anti-leukemia cell agent comprising a polyphenol comprising t or Sangiin H-6 as an active ingredient. 2. An anti- leukemic cell agent comprising cinnamon-derived procyanidin C-1 as an active ingredient.