JPH06135845A - B cell function inhibitor - Google Patents

Abstract

PURPOSE:To obtain a medicine having action useful for treating autoimmune disease, especially having inhibitory action on function of B cell. CONSTITUTION:A b cell function inhibitor comprises KEISHIBUSHITOU known to be useful for symptoms such as ague or fever as an active ingredient. KEISHIBUSHITOU has a blending range of 4.0-6.0 KEISHI (bark of Cinnamomum cassia), 0.5-3.0% BUSHI (tuber of Aconitum carmichaeli, 1.0-3.0 ginger, 3.0-12.0 DAISOU (fruit of Zizyphus jujuba) and 2.0-3.0 KANZOU (root of Glycyrrhiza glabra), this KEISHIBUSHITOU used directly as it is or its extract, preferably an extract with water is combined with a well-known carrier for medicine, pharmaceutically manufactured, used and is effective for treating rheumatoid arthritis, autoimmune spherical hepatitis, autoimmune hemolytic anemia, autoimmune strumitis and other autoimmune diseases. The weight of the active ingredient is 3-15g per adult daily and properly used dividedly several times daily.

Description

Detailed Description of the Invention

[0001]

INDUSTRIAL APPLICABILITY The present invention has few side effects and is useful for treating rheumatoid arthritis, autoimmune glomerulonephritis, autoimmune hemolytic anemia, autoimmune thyroiditis and other autoimmune diseases. The present invention relates to a drug having a B cell function suppressing action.

[0002]

2. Description of the Related Art Immunity is a mechanism that protects the living body from invasion from the inside and outside, and bone marrow-derived lymphocytes, T cells and B cells, play a major role. T cells and B cells are responsible for humoral immunity and cellular immunity, respectively, and the function of B cells (humoral immunity) is particularly important. For example, in many of the autoimmune diseases represented by rheumatoid arthritis, autoimmune glomerulonephritis, autoimmune hemolytic anemia, autoimmune thyroiditis, and others, regulatory T cells that suppress or enhance B cell function are provided. , And an increase in the number of antibodies against autologous biogenic components produced by B cells.

[0003] In general, the drugs effective as therapeutic agents for autoimmune diseases include, for example, corticosteroids, immunosuppressants, and platelet aggregation inhibitors, all of which have a definitive therapeutic effect. Not really. Moreover, many of them are known to have serious side effects, and particularly, when an immunosuppressive drug is administered in large amounts for a long period of time, the side effects almost certainly develop.

Therefore, it has been desired to develop a drug having few side effects and a useful action for treating an autoimmune disease, particularly an action of suppressing B cell function.

[0005]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, it has been found that Keishibushito has an action of selectively inhibiting the function of B cells (B cell function inhibiting action). Furthermore, they have a therapeutic effect on rheumatoid arthritis, which is an example of an autoimmune disease, and have completed the present invention.

That is, the present invention contains B which contains Keishibushito (hereinafter referred to as the active ingredient of the present invention) as an active ingredient.
It is a cell function inhibitor.

[0007] The prescription of Keishibushi-to, which is the active ingredient of the present invention, is described in the classical Chinese herbal formula (Kinbyo, etc.),
Although there are some differences, the compounding range of the crude drug is generally as follows. Further, Keishibushito is known to be used for symptoms such as chills and fever, but it has never been known that it has a B cell function suppressing effect and is useful for treating autoimmune diseases. That was not done.

Keishibushikoyu Keishi 4.0-6.0 Fuzi 0.5-3.0 Ginger 1.0-3.0 Ohju 3.0-12.0 Licorice 2.0-3.0 As the active ingredient of the invention, Keishibushito of the above formulation may be used as it is, or an extract thereof may be used as an active ingredient in combination with a known pharmaceutical carrier to prepare a pharmaceutical preparation.

Examples of the extract of Keishibushi-to include various aqueous solvent extracts of Kei-shibushi-to, and it is preferable to use a water extract. As a specific example of adjusting the Keishibushito extract, there is a method of extracting Keishibushito of the above composition with 10 times the amount of hot water and filtering the resulting extract. This extract can be dried if necessary and used as a dry powder.

Next, the production of the active ingredient of the present invention will be described in detail with reference to specific examples.

SPECIFIC EXAMPLE 1 Katsura 4.0 g, Bushi 1.0 g, Ginger 1.0 g, Ojumu 3.
0g and licorice 2.0g mixed crude drug (Keishibushito: 11
110 g of purified water was added to g), and the mixture was heated and extracted at 100 ° C. for 1 hour. The obtained extract was filtered and then spray-dried to obtain 2.5 g of dry extract powder.

Specific Example 2 800 g of katsura, 200 g of konjac, 200 g of ginger, and 60 otsuki
Mixed herb of 0g and licorice 400g (Shosaikoto: 2.2k
22 g of purified water was added to g), and the mixture was heated to 100 ° C. and extracted for 1 hour. The obtained extract was centrifuged and the residue was separated to obtain 20 l of a solution.

This solution was subjected to aseptic clarification with a 0.3 μm membrane filter (manufactured by Toyo Roshi Kaisha, Ltd.). The obtained filtrate was ultrafiltered using a diafilter G-10T (manufactured by Bio Engineering Co., Ltd .: molecular weight cut off 10,000). For this ultrafiltration, a membrane with a diameter of 152 mm was set on the lower surface of a container with an internal volume of 2.0 l and the pressure was 3 kg / cm ^2.
Then, as the liquid in the container was concentrated, about 2 l of purified water was added. As a result, 20 l of ultrafiltrate was obtained.

Next, experimental examples will be described to explain that the active ingredient of the present invention has a B cell function suppressing action and further has a therapeutic effect on rheumatoid arthritis which is an example of an autoimmune disease.

Experimental Example 1 Proliferation reaction by mitogen Immunization and administration method of test sample Eight-week-old female Lewis strain rat corresponds to human 10 times amount 5
00 mg / kg of the active ingredient of the present invention obtained in Example 1 was orally administered, and 7 days after the administration, C. immunization was performed. C ◆ Immunization was a bovine-derived type dissolved in 0.1 M acetic acid to a concentration of 1 mg / ml. ◆ An equal amount of incomplete Freund's adjuvant was added to a collagen solution, and 1 ml each was made into an emulsion with a thermo-mixer. This was done by subcutaneous injection at the ridge. C ◆ After immunization, the animals were raised according to the following classification, and the action of the active ingredient of the present invention was examined. Group A: Water was administered for the entire period. Group B: Keiishibushito obtained in Example 1 was administered for the entire period. Group C: Keishibushito obtained in Example 1 was administered from the day of onset.

Adjustment of mitogen LPS (lipopolysaccharide) as a B cell mitogen, ConA (concanavalin A) and PHA (phytohemagglutinin) as 10% F as cell mitogens.
1 mg / m in RPMI1640 medium with CS (fetal calf)
After lysing to a volume of 1 and sterilizing it with a 0.22 μm filter, LPS was 200 μg / ml, ConA
And PHA were adjusted to be 20 μg / ml. In addition, the collagen solution (adjusted to pH 6.0 with sodium hydroxide) was sterilized with a 0.45 μm filter and RPMI1
It was adjusted to 200 μg / ml in 640 medium.

Preparation of Cell Suspension C ◆ On day 10 after immunization, the rats of group A and group B were decapitated, the blood was removed from the femur, and the cold RPMI1640 was cut.
Immersed in medium. Remove the attached muscle with a scalpel,
Both ends of the bone were cut with scissors to expose the bone marrow cavity. Put 2 ml of RPMI1640 medium into a Petrai 60M / M Petri dish, dip one end of the bone, and 20G on the other end.
Bone marrow cells were extruded with RPMI 1640 medium in 8 ml. The bone marrow cells were loosened and suspended in 10 ml of RPMI1640 medium supplemented with FCS with tweezers or the like. Centrifuge the cell suspension with a Kubota centrifuge (1000 rp
m), remove the supernatant, and remove 10 ml of RPMI164
After washing with 0 medium, centrifugation (1000 rpm, 5 minutes),
The supernatant was removed. It was suspended again in 5 ml of RPMI1640 medium, counted and adjusted with RPMI1640 medium so as to be 1 × 10 ⁶ cells / ml.

Mitogen reaction A 96-well plate (FALCON 3075) was added to each mitogen, collagen solution or FCS-added RP.
100 μl of MI1640 medium was added according to the following classification, and 100 μl of cell suspension was added. 48 using a carbon dioxide incubator (5% carbon dioxide, 37 ° C)
5m after 0.22μm filter sterilization
g / ml MTT [3- (4,5-dimethylthiazol-
2 yl) -2,5-diphenyl tetrazolium bromide] PBS solution was added in an amount of 20 μl each, and the mixture was further cultured in a carbon dioxide incubator for 4 hours. After culturing, 1%
100 μl of SDS (sodium dodecyl sulfate) was added and left at room temperature. Approximately 12 hours later, 57 with a model 450 microplate reader (manufactured by Bio-Rad)
Absorption at 7 nm was measured (OD = OD ₅₇₇ −OD
₆₅₅ ). The results were obtained as a ratio with the absorption value of the normal group being 1. The results are shown in Table 1. LPS group: Add LPS ConA group: Add ConA PHA group: Add PHA Collagen group: Add collagen Normal group: Add RPMI1640 medium with FCS

Table 1 Proliferative response to mitogen

Experimental Example 2 Type ◆ Measurement of antibody titer to collagen Collection of serum Stomachs of the rats were cut off immediately before immunization of the above-mentioned C of groups A, B and C, and centrifuged with biofuge A (13000 r).
pm, 5 minutes) and the serum was collected. This was used as the serum on day 0, and thereafter, serum on days 7, 14, 21, and 28 was collected.

Measurement PB was applied to a 96-well enzyme-labeled immunosorbent assay multiplate.
A collagen solution diluted to 5 μg / ml with S was added to each plate in an amount of 100 μl for adsorption. (4 ° C., overnight) The collagen solution was discarded, and 200 μl of 1% BPA (albumin and borin composition was dissolved in PBS) was applied to the collagen-unadsorbed solid surface. After leaving it at room temperature for 30 minutes, it was discarded and washed with PBS containing 0.05% Tween 20 three times. Then, added serum diluted 2 ^9-fold by limiting dilution in PBS by 50 [mu] l, and allowed to stand for one hour at room temperature. After discarding this and washing 3 times with PBS containing Tween 20, 50 μl of peroxidase-rabbit anti-rat IgG or peroxidase-rabbit anti-rat IgM diluted 4000 times with PBS was added to each. After leaving it at room temperature for 1 hour, it was washed with PBS containing Tween 20 three times, and an o-PDA (phenylenediamine) solution [o-PDA was used as a substrate.
0.1 M citric acid to 0.4 μg / ml
It was dissolved in a 0.2 M phosphate buffer (pH 5.0), and immediately before use, 100 μl of 30% hydrogen peroxide solution was added at a ratio of 2.778 μl / 25 ml). After leaving it for 30 minutes at room temperature in the dark, 25 μl of 6N sulfuric acid was added to stop the reaction. Absorption was measured at 490 nm on a model 450 microplate reader. The results are shown in Tables 2-5.

Table 2 IgG against collagen (1 /
2)

Table 3 IgG against collagen (2 /
2)

Table 4 IgM for collagen (1 /
2)

Table 5 IgM for collagen (2 /
2)

Experimental Example 3 Effect on Chronic Arthritis The rats of Group A, Group B and Group C were observed according to the following criteria up to 30 days, starting from the day of immunization with C ◆. As a result, an incidence rate and an average score (2 limbs per animal) with the day of immunization as 0 day and an average score with the day of onset as 0 day were obtained. The results are shown in Tables 6-10. 0: Not presenting 1: Redness and slight swelling of the ankle etc. 2: Redness and swelling of the ankle etc. 3: Redness and swelling of the middle root part 4: Foot shank All, redness and severe swelling

Table 6 Incidence rate

Table 7 Mean score of onset state based on the day of immunization (1/2)

Table 8 Mean score of onset state based on the day of immunization (2/2)

Table 9 Average score of the onset state based on the day of onset (1/2)

Table 10 Average score of onset state based on the day of onset (2/2)

As is clear from the above results, the administration groups (groups B and C) of the present invention have a significant B cell function inhibitory action as compared with the non-administration group (group A), and further, autoimmunity. It was confirmed to have a therapeutic effect on rheumatoid arthritis, which is an example of a sexually transmitted disease. Therefore, Keishibushito, which is the active ingredient of the present invention, is useful as a B cell function inhibitor.

Keishibushi-to, which is the active ingredient of the present invention, has a long history as a Chinese medicine and its safety has been confirmed, so it can be safely used. For example, for mice and rats, the limit dose is 15 g /
It is extremely safe, as evidenced by the fact that no cases of death were observed after oral administration of kg.

Next, the dose and formulation of the active ingredient of the present invention will be described.

The dosage form of the active ingredient of the present invention is not particularly limited and may be appropriately selected and used as required. Oral preparations such as tablets, capsules, granules, fine granules and powders, injection preparations, Parenteral agents such as suppositories may be mentioned.

In order to exert the intended effect, it depends on the age, body weight and degree of disease of the patient, but usually 3 to 15 g as the weight of the active ingredient of the present invention is divided into several times a day in adults. Seems to be appropriate.

The active ingredient of the present invention includes tablets, capsules,
Oral preparations such as granules are produced by a conventional method using starch, lactose, sucrose, mannitol, carboxymethyl cellulose, corn starch, inorganic salts and the like.

In this type of preparation, a binder, a disintegrating agent, a surfactant, a surfactant, a fluidity promoter, a flavoring agent, a coloring agent, a fragrance, etc. may be appropriately used in addition to the above-mentioned excipients. You can
Specific examples of each are as shown below.

[Binder] Starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch,
Methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, crystalline cellulose, ethyl cellulose, polyvinylpyrrolidone, macrogol.

[Disintegrant] Starch, hydroxypropyl starch, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, carboxymethyl cellulose, low-substituted hydroxypropyl cellulose.

[Surfactant] sodium lauryl sulfate,
Soy 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.

[Fluidity promoter] Light anhydrous silicic acid, dried aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate.

The active ingredient of the present invention can also be administered as a suspension, emulsion, syrup or elixir, and these various dosage forms contain a flavoring agent and a coloring agent. Good.

On the other hand, parenteral preparations are manufactured by a conventional method,
Generally, distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol and the like can be used as the diluent. Further, if necessary, a bactericide, a preservative, and a stabilizer may be added. Further, from the viewpoint of stability, this parenteral preparation may be filled in a vial or the like and then frozen, the water content may be removed by a usual freeze-drying technique, and a liquid preparation may be re-prepared from the freeze-dried product immediately before use. Further, an isotonicity agent, a stabilizer, a preservative, a soothing agent and the like may be added as needed.

Next, the present invention will be described in more detail by showing Examples of preparations of the active ingredient of the present invention, but the present invention is not limited thereto.

Example 1 Corn starch 21 g Crystalline cellulose 10 g Carboxymethyl cellulose calcium 7 g Light anhydrous silicic acid 1 g Magnesium stearate 1 g Keishibushi-to obtained in Example 1 160 g Total 200 g It was compression molded with a tableting machine to obtain 200 mg tablets. This tablet contains 160 mg of Keishibushito obtained in Example 1, and 20 to 80 tablets for adults are divided into several times and taken.

Example 2 Corn starch 29 g Magnesium stearate 2 g Carboxymethyl cellulose calcium 8 g Light anhydrous silicic acid 1 g Keishibushi-to obtained in Example 1 160 g Total 200 g According to the above formulation, After being compression-molded by, the mixture was crushed by a crusher and sieved to obtain granules. 1 g of this granule contains 800 mg of Keishibushito obtained in Example 1, and 4 to 18 times a day for adults.
Take g in several divided doses.

Example 3 Cornstarch 19 g Light anhydrous silicic acid 1 g Keishibushi-to obtained in Example 1 180 g Total 200 g
Was filled in a No. 2 capsule. One capsule of this capsule contains 20 mg of Keishibushito obtained in Example 1, and 20 to 80 capsules for an adult are divided into several times and taken.

Example 4 To 20 liters of Keishibushito obtained in Example 2, 300 g of alanine (free of heat-generating substance) was added, dissolved and freeze-dried. This lyophilized product was dispensed into 900 vials to obtain an injection. 1 vial of this injection contains lyophilized product 406
It contained mg and was easily dissolved in 10 ml of purified water. In addition, the injectable solution after dissolution had a transmittance of 92% (550 nm) and passed the test method for pyrogenic substances of the Japanese Pharmacopoeia.

Claims (1)

[Claims] 1. A B cell function inhibitor containing Keishibushito as an active ingredient.