WO1995030413A1

WO1995030413A1 - Hematopoietic stem cell proliferation accelerator

Info

Publication number: WO1995030413A1
Application number: PCT/JP1994/001050
Authority: WO
Inventors: Haruki Yamada; Hiroaki Kiyohara; Masumi Hirano; Susumu Ikehara; Hideki Kawamura; Norito Takemoto
Original assignee: The Kitasato Institute; Tsumura & Co.
Priority date: 1994-05-10
Filing date: 1994-06-29
Publication date: 1995-11-16
Also published as: AU7082694A

Abstract

A hematopoietic stem cell proliferation accelerator containing at least one fatty acid such as palmitic, stearic, petroselaidic or elaidic acid as the active ingredient. Because these acids have an excellent activity of proliferating hematopoietic stem cells and a high safety, they can prevent hematopoietic stem cell cytopenia due to diseases and as the side effect of administration of anticancer drugs, radiation therapy, and so forth, thus being extremely advantageous for treating or preventing these anomalies.

Description

Disclosure of the invention

The present inventors found that Juzentaihoto, which is a Kampo medicine used mainly for post-illness or post-operative physical weakness and anorexia, has the effect of increasing the number of pluripotent hematopoietic stem cells. As a result of further investigation of the active ingredient for its action, it was found that the saturated fatty acid and unsaturated fatty acid contained in the preparation are the active substance. Further, they have found that the action can be recognized not only in fatty acids contained in Juzentaihoto but also in other fatty acids, and thus completed the present invention.

That is, the present invention provides a hematopoietic stem cell proliferating agent containing one or more fatty acids as active ingredients. BEST MODE FOR CARRYING OUT THE INVENTION

In the hematopoietic stem cell proliferating agent of the present invention, the fatty acid used as an active ingredient may be fractionated and purified from Juzentaihoto, but it may be present in other natural products. However, it is also possible to use a commercially available product that is separated and purified as a single product from these. The fatty acid used in the present invention may be saturated or unsaturated, and its carbon number is preferably in the range of 4 to 24, particularly in the range of 16 to 18. Further, the unsaturated degree of the unsaturated fatty acid is preferably in the range of 1 to 6, particularly in the range of 1 to 3.

Specific examples of fatty acids include the following.

[Saturated fatty acid]

Carbon number; number of double bonds

Palmitic acid 1 6

Stearic acid 1 8

¾ acid 4

Cabronic acid 6

Power Prilic acid 8

Power Puric acid 10

Behenic acid 2 2 <1>

Clear

Hematopoietic stem cell proliferation agent Technical field

The present invention relates to a hematopoietic stem cell proliferating agent that promotes proliferation of pluripotent hematopoietic stem cells, and more specifically, reduction of hematopoietic stem cells due to infection or malignant tumor, hematopoietic stem cells due to side effects such as anticancer agents, immunosuppressive agents, and radiation. The present invention relates to a hematopoietic stem cell proliferating agent that prevents or treats a decrease in blood pressure. Background technology

As chemotherapy for malignant tumors, development of new anti-cancer agents, multi-drug combination therapy combining anti-cancer agents with different action mechanisms, and active chemotherapy in addition to surgical therapy are being performed. Tumor shrinkage and prolongation of survival time have been recognized by these advances in chemotherapy, but on the other hand, side effects from anticancer agents have become a problem, which is a major obstacle to cancer treatment.

For example, mitomycin C, which is one of the typical anticancer agents in chemotherapy, has a strong anticancer activity, but its use is restricted because it exhibits leukopenia and thrombocytopenia.

In order to reduce such side effects, picibanil and various colony stimulating factors

(colony-stimulant factor (CSF)) is used, but these compounds have another side effect problem, and because they are administered by injection, they are not easy to administer at home. Was left.

Therefore, it has been desired to develop a drug that not only has an excellent hematopoietic stem cell proliferating action but can be orally administered and is highly safe. Unsaturated fatty acids such as acids Can be mentioned.

Although hematopoietic stem cells are originally capable of self-renewal and differentiate into blood cells, the hematopoietic stem cell proliferating agent of the present invention is a drug that acts on this hematopoietic stem cell to promote proliferation and differentiation of hematopoietic stem cells. Is called. Therefore, the hematopoietic stem cell proliferating agent of the present invention is an index for a drug used for the purpose of preventing the reduction of hematopoietic stem cells due to various causes and the proliferation of hematopoietic stem cells, and is effective for the treatment of the following diseases, for example: It includes various medicines.

Infection

Rubella, measles virus, lichen, tuberculosis, sarcoidosis, coccidioidomycosis, AIDS, Cytomegalovirus, E-B virus etc.

Malignant tumor

Cancer, sarcoma, leukemia, malignant lymphoma, etc.

Autoimmune disease

Systemic lupus erythematosus, rheumatoid arthritis, dermatomyositis, scleroderma etc. Protein loss disease

Nephrotic syndrome, protein-losing enteritis

Other

Diabetes, alcoholic cirrhosis, malnutrition, burns, splenectomy, sickle cell anemia, uremia, subacute sclerosing panencephalitis, anti-cancer drug Reduction of

To prepare the hematopoietic stem cell proliferating agent of the present invention, the active ingredient, fatty acid, is formulated as it is or with a conventional pharmaceutical carrier to prepare tablets, capsules, soft capsules, granules, fine granules, powders, Oral preparations such as liquid preparations and syrups, or non-seed preparations such as injectable preparations and drip preparations may be used.

In order to exert the intended effect of the fatty acid used in the present invention by the administration of cotton, it depends on the patient's age, body weight, and degree of disease. It is considered appropriate to administer 1 O mg to 10 g as several divided doses daily. <3>

Undecanoic acid 1 1

Lignoceric acid 24

[Unsaturated fatty acid]

Carbon number Number of double bonds

Petroselaidinic acid 18

Elaidic acid 1 8 1

Linoleic acid 1 8 2

(Linoelaidic acid)

Trans-vaccenic acid 18

Vaccenic acid 18

Hiichirinoleic acid 1 8 3

Arinoleic acid 1 8 3

Petroselinic acid 18

Oleic acid 1 8 1

Linoleic acid 1 8 2

Arachidonic acid 2 0 4

Eicosapentaenoic acid 205

Docosahexaenoic acid 2 2 6

L acid acid 2 2 1

Myristic acid 1 4

(Myristoleic acid)

Palmitoleic acid 1 6

(Palmitoleic acid)

Cis-1 0-Nonadecenoic acid 1 9

5-Eicosenoic acid 20 Of these, preferred are the saturated fatty acids of undecanoic acid, behenic acid, palmitic acid, and stearic acid, and oleic acid, linoleic acid, and petroselide. Depending on the patient's age, body weight, and degree of illness, the usual adult dosage is 0.5 mg to 1 g daily of the active ingredient administered by intravenous injection, intravenous drip infusion, subcutaneous injection, intramuscular injection, etc. It seems appropriate to administer

This parenteral preparation is also manufactured according to a conventional method, and is generally used as a diluent in distilled water for injection, saline solution, aqueous glucose solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol. Etc. can be used. Further, if necessary? A δ-bacterial agent, a preservative, and a stabilizer may be added. In addition, from the viewpoint of stability, this parenteral preparation can be frozen after filling in vials, etc., water can be removed by the usual freeze-drying technique, and the liquid preparation can be reconstituted from the freeze-dried product immediately before use. .. In addition, if necessary, suitable II, isotonicity agent, stabilizer, preservative, soothing agent, etc. may be added.

The hematopoietic stem cell proliferating agent of the present invention can be used not only alone but also as a mixture by combining it with other anti-cancer agents and the like.

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

Example 1

Bone marrow cell proliferation effect:

MS-5 cells derived from C 3 H mouse bone marrow, which is a hematopoietic feeder cell line, were subjected to 96-well micro-mouth plating with 10% horse serum-containing ΜΕΜα-medium (with 10 Μ of hydrated cortisone). After the seedlings were grown to cover the entire surface, the growth was stopped by irradiation with 20 G y of ^e °C o. Next, this cell as a feeder layer, implantation 1 0 ⁴ to Ri hematopoietic stem cells of mice partially purified per each Ueru, added to 4 each test substance in various concentrations - cultured for 5 days, ³ H — Thymidine incorporation was measured.

^{From the 3} H-thymidine incorporation amount, the cell growth rate was calculated by the following formula. The results are shown in Table 1.

Uptake of cells containing test substance

Cell proliferation rate (%) = 100 0 Uptake of cells without test substance <5>

It

To prepare solid preparations among the lotus agents, the active ingredients such as fatty acid and starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, inorganic salts, etc. are prepared according to the usual method. It may be combined with an effervescent agent.

In addition to the above-mentioned excipients, the oral preparations include binders, disintegrants, surfactants, lubricants, flow promoters, flavoring agents, coloring agents, flavors, etc., as exemplified below. It can be used as appropriate.

[Binder]

Starch, Dextrin, Gum arabic, Gelatin, Hydroxypropyrus starch, Methylcellulose, Carboxymethylcellulose sodium, Hydroxypropylcellulose, Crystalline cellulose, Ethylcellulose, Polyvinylpyrrolidone, Macrogol ..

[Disintegrant]

Starch, hydroxypropyl starch, carpoxymethyl cellulose sodium, carpoxymethyl cellulose calcium, carboxymethyl cellulose, low-substituted hydroxypropyl cellulose.

[Surfactant]

Sodium lecithin, soybean lecithin, sucrose fatty acid ester, polysorbate 80, polyoxyethylene hydrogenated castor oil.

[Lubricant]

Talc, waxes, hydrogenated vegetable oil, polyethylene glycol.

[Fluidity promoter]

Light anhydrous carboxylic acid, dried aluminum hydroxide gel, synthetic aluminum gayate, magnesium silicate.

Further, the hematopoietic stem cell proliferating agent of the present invention can be made into a soft capsule by a conventional method after the active ingredient is used alone or in combination with other oral oil preparations. In addition, liquid oral preparations such as! 1 Suspensions, emulsions, syrups, and elixirs may be added, and these preparations may be mixed with a flavoring agent and a coloring agent.

On the other hand, in order to exert the hematopoietic stem cell proliferation action of fatty acids by parenteral administration, Table 1 (continued) Fatty acid 1 Concentration Breeding rate

j (/g/m 1) (% )palmitoleic acid 丄 .u 丄 7

Cis-1 0—Nonadecenoic acid 0 • 5 1 2 4

〃 1 • 0 1 2 4

5-Eicosenoic acid 0 5 1 2 1

〃 1 • 0 1 2 8 ；Behenic acid! 0 5 1 5 9

1 0 1 3 7

"\

L acid acid i 0 5 1 2 3

// I 1 0 1 3 5

Docosahexaenoic acid 0 5 1 2 4

// 1 0 1 6 0

Lignoceric acid 1 0 1 2

⁴ As is clear from this result, the proliferation of hematopoietic stem cells was increased by the addition of fatty acid.

Example 2

Hematopoietic stem cell proliferation effect:

The proliferative effect of the compounds of the present invention on hematopoietic stem cells was examined by the mouse spleen colony method. That is, mitomycin C (MMC, 1 mg/kg) was intraperitoneally administered to each group of 4 C57B LZ6 mice (male, 8 weeks old) once a day for 7 consecutive days. In the group of the present invention, administration of M-MC was started at the same time as l%Tween 80, and oleic acid with S turbidity was started, and was orally administered once a day, 6 times a week for 3 weeks until the end of the experiment.

Two weeks after the end of MMC administration, bone marrow cells were obtained from the mouse femur using Hanks' solution. <7> Result:

Table 1 Fatty acid concentration increase rate

(z g / m 1) (%) Petroselaidinic acid 0.5 1 7 5 〃 1-0 1 6 2 Elaidic acid 0.2 1 6 8 No / 0.5 1 7 0

1.0 1 8 2 Oleic acid 0.1 1 6 6

// 0 .5 2 0 5 linoleic acid 0 .2 1 3 5 hii linolenic acid 0 .1 1 5 3 〃 0 .2 1 6 3 〃 0 .5 2 2 3 arynoleic acid 0.2 1 7 Petroselinic acid 1.0 1 6 3 Palmitic acid 0.5 1 5 4 Steariic acid 0.5 1 6 5 Undecanoic acid 0.5 1 3 6 〃 1.0 1 3 6 Myrist Rayonic acid 0.5 1 2 5 // 1 .0 1 2 4 ① Dextrin 40 g

② Crystalline cellulose 20 g

③ Carboxymethyl

Cellulose calcium 5 g

④ Polyethylene glycol

6 0 0 0 5 g

⑤ Oleic acid 30 g Total 100 g

[Production method]

According to the above prescription, (1) to (5) were uniformly mixed and compression-molded with a tableting machine to obtain 100 mg tablets.

Each tablet contains 3 mg of oleic acid, and 1 to 20 tablets for adults should be taken in several divided doses daily.

Example 4

Tablets:

A hematopoietic stem cell proliferating agent (tablet) was manufactured by the following formulation and manufacturing method.

[Direction]

① Dextrin 79 g

② 10% Hydroxy Pro Pill

Cellulose ethanol solution 50 g

③ Carboxymethyl

Cellulose calcium 5 g

④ Polyethylene glycol

6 0 0 0 1 g

⑤ Linoleic acid 10 g Total 45 g

[Production method] <9>

A suspension was prepared. This bone marrow cell suspension was subjected to hemolysis treatment of erythrocytes with 0.83% ammonium chloride buffer, the cells were washed, the cell number was measured with a Coulter counter, and 8 days after cell transfer, for measurement, 3 X 1 0 ⁵ mice, the measurement after cell transfer 1 04, to adjust the number of cells to be 1.5 X 1 0 ⁵ Z animals.

The bone marrow cell preparation prepared above was transferred from the tail vein to C57BL6 mice that had been irradiated with X-rays. The spleen was extracted 8 days after cell transfer (colony forming unit in spleen; CFU-S, day8) and 14 days after (CFU-S, day14). After measuring the spleen weight, it was stained with Bouin's staining solution and the number of colonies was measured. The increase rate of CFU-S was calculated by the following formula. The results are shown in Table 2.

A-B

Proliferation rate of hematopoietic stem cells (%) =

B

A: Number of colonies in MMC and oleic acid combination administration group (invention group)

B: Number of colonies in MMC single administration group (comparison group)

Oleic acid concentration Hematopoietic stem cell proliferation rate (%)

i.m k g)

8 days later 14 days later

1 3 4 4 1 63

1 0 2 1 3 1 94 As is clear from this result, hematopoietic stem cells were proliferated by the addition of oleic acid, Example 3

Tablets:

A hematopoietic stem cell proliferating agent (tablet) was manufactured according to the following formulation and manufacturing method.

[Direction] Cellulose ethanol solution 35 g

③ Arinolenoic acid 10 g Total 1 3 1.5 g

[Production method]

According to the above prescription, (1) to (3) were evenly warmed and mixed. After granulating with an extrusion granulator, it was dried and separated by IS to obtain granules.

This granule l g contains linolenic acid l O O mg, and an adult should take 0.3 to 6 g daily in divided doses.

Example 7

Capsule ：

A hematopoietic stem cell proliferating agent (capsule) was manufactured by the following formulation and manufacturing method.

[Direction]

① Anhydrous crystalline maltose 89.5 g

② Light caylic anhydride 0.5 g

③ Palmitic acid 10 g Total 100 g

[Production method]

According to the above prescription, (1) to (3) were uniformly mixed, and 20 O mg was filled into No. 2 capsule.

1 capsule of this capsule contains 20 mg of palmitic acid, and 1 to 30 capsules per adult should be taken in several divided doses per day.

Example 8

Injection:

A hematopoietic stem cell proliferating agent (injection) was manufactured by the following formulation and manufacturing method.

[Direction]

① Distilled water for injection 8 9.5 g

② Soybean oil 5 g <11>

According to the above prescription, mix ①, ② and ⑤ uniformly, blend by a conventional method, granulate by an extrusion granulator, dry and crush, then mix ③ and ④. Then, compression molding was performed using a tableting machine to obtain 300 mg tablets.

Each tablet contains 3 O mg of linolenic acid, and 1 to 20 tablets for adults are to be taken in several divided doses.

Example 5

Condylar agent:

A hematopoietic stem cell proliferating agent (granules) was manufactured by the following formulation and manufacturing method.

[Direction]

① Dextrin 8 2 g

② Polyethylene glycol

6 0 0 0 2.5 g

③ Carpoxymethyl

Cellulose calcium 5 g

④ Light anhydrous caic acid 0.5 g

⑤ Petroselidenic acid 10 g Total 100 g

[Production method]

According to the above prescription, (1) to (5) were uniformly mixed, compression-molded with a compression molding machine, crushed with a crusher, and sieved to obtain granules.

This granule l g contains 1 O O mg of petroselaidinate, and 0.3 to 6 g for adults is to be taken in divided doses.

Example 6

Condylar agent:

[Direction]

① Dextrin 8 6.5 g

② 10% Hydroxypropyl

ADAACCCCCDBBBBCCBBBC (5 7) Summary

MUTEGYGHNEKABFRFZ JI

A hematopoietic stem cell proliferating agent containing one or more kinds of fatty acids such as palmitic acid, stearic acid, petroceredic acid, and elaidic acid as active ingredients is disclosed. The fatty acid used in the present invention has an excellent hematopoietic stem cell-proliferating action and is highly safe. It prevents the decrease of hematopoietic stem cells and can be used advantageously in the extreme treatment and prevention of these. MMMMMMMMNNNNRP LLLLPL WUOT KDXORTVGNRECZLLL

Lurumumamomomomomomo nibobo mezulanoriri

Beruru Luna

-Semaagashin Uralovekouuriritodo

Lupa Ivanigas Dan

Ruruaani Power Bukazido

Rug

Do

For informational use only

Codes used to identify PCT countries in international pan-fretted international applications published under the PCT Armenia EE Estonia RU Russia Fast Country Austria Spain Spain Spain Sweden Australia FI Finland SE Su! 1 Den Barbados FR France SG Singapore Belgium GA Gabon SI Slovenia Bulgina Faso GB United Kingdom SK Slovak Republic Bulgaria GE Georgia SN Senegal

G Guinja SZ Swaziland Budir GR Greece TD Chard Belarus HU Hungary 1 TG Togo Canada IE Ireland TJ Tajikistan Central African Republic IS Iceland T Tortame-sta Congo IT Italy TT Trinidad ■ Tobagosys JP Japan UA Utari *National KE Kenya UG Uganda Cameroon KG Kyrgyzstan us Country

China KP (Democratic People's Republic of Korea UZ Republic of Chebe Republic of KR KR Greater Republic V Vietnam Vietnam KZ Kazakhstan

Denmark I I Liechtenstein

Claims

The scope of the claims

1. A hematopoietic stem cell proliferating agent containing one or more fatty acids as active ingredients.

2. The hematopoietic stem cell proliferating agent according to claim 1, wherein the fatty acid is a fatty acid having 4 to 22 carbon atoms.

3. The hematopoietic stem cell proliferation agent according to claim 1 or 2, wherein the fatty acid is a saturated fatty acid.

4. The hematopoietic stem cell proliferating agent according to claim 1 or 2, wherein the fatty acid is an unsaturated fatty acid.

5. The hematopoietic stem cell proliferation according to claim 3, wherein the fatty acid is selected from palmitic acid, stearic acid, butyric acid, cabronic acid, erythroglyceric acid, cabrinic acid, behenic acid, undecanoic acid or lignoceric acid. Agent.

6. Fatty acids are petroselaidic acid, elaidic acid, linoelaidic acid. trans-pacenoic acid, paxenoic acid, arynolenoic acid, α-linolenic acid, petroceric acid, Oleic acid, linoleic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, erucic acid, myristoleic acid, palmitoleic acid, cis-1 10 -The hematopoietic stem cell proliferating agent according to claim 4, which is selected from nonadecenoic acid and 5-eicosenoic acid.

7. The hematopoietic stem cell proliferating agent according to any one of claims 1 to 6 for the purpose of preventing/treating hematopoietic stem cell depletion caused by infectious diseases, malignant tumors, autoimmune diseases or protein loss diseases ..

8. The hematopoietic stem cell proliferating agent according to any one of claims 1 to 6, which is intended to reduce side effects of an anticancer agent, an immunosuppressive agent, or radiation. <13>

③ Soybean phospholipid 2.5 g

④ Glycerin 2 g

⑤ Linoleic acid 1 Total 100 g

[Production method]

According to the above prescription, (5) was dissolved in (2) and (3), and the solutions of (1) and (4) were added and emulsified to obtain an injection. Industrial availability

The fatty acid used in the present invention has an excellent hematopoietic stem cell proliferating action as shown in the above test examples.

All of these fatty acids are known to be contained in foods.For example, no fatal cases were observed after oral administration of 1 to 100 mg kg to rat for 3 consecutive weeks. As is clear from the above, the safety is high.

Therefore, according to the hematopoietic stem cell proliferating agent of the present invention, it is possible to safely reduce the number of hematopoietic stem cells due to various infectious diseases, malignant tumors, and other diseases, and to administer anticancer agents, steroid drugs, and side effects such as irradiation. Since it is possible to prevent the reduction of hematopoietic stem cells and to proliferate hematopoietic stem cells, it is extremely advantageous in treating and preventing these diseases and side effects.