JP2001348342A - Immunomodulating and antiinflammatory agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a pharmaceutical composition for new application, comprising histamine added γ-globulin as an active ingredient. SOLUTION: This pharmaceutical composition such as an immunomodulating and antiinflammatory agent comprises histamine-added γ-globulin as an active ingredient. Since the pharmaceutical composition has immunomodulating actions definitely different from those of a conventional immunosuppressive agent, the composition is useful as a therapeutic agent for diseases in which the immune system undergoes an abnormality such as autoimmune diseases, e.g. rheumatoid arthritis, systemic lupus erythematosus or multiple sclerosis or various kinds of immunodeficiency syndrome. The pharmaceutical composition has inhibitory actions on eosinophilia, can be used as an excellent antiinflammatory agent and has ultrahigh usefulness.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to a novel pharmaceutical use of histamine-added γ-globulin, and more particularly to histamine-added γ-globulin.
The present invention relates to a pharmaceutical composition containing globulin as an active ingredient, such as an immunomodulator and an anti-inflammatory agent.

[0002]

BACKGROUND ART A complex of γ-globulin and histamine is known as a histamine-added γ-globulin preparation,
It has the effect of restoring the reduced histamine fixation ability in allergic patients and asthmatic patients, and as a nonspecific desensitizing therapeutic agent, bronchial asthma, allergic rhinitis, urticaria, chronic eczema, atopic dermatitis, etc. It is used for the treatment of allergic skin diseases. This drug has also been found to have an inhibitory effect on histamine release, and has no side effects such as antihistamines or corticosteroids used as symptomatic drugs, and is widely used as a highly safe drug. I have. The present inventors have conducted intensive studies on this histamine-added γ-globulin, and as a result, have found a novel pharmacological action of an immunomodulatory action and an inhibitory action on eosinophilia, and completed the present invention.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel pharmaceutical composition containing histamine-added γ-globulin as an active ingredient.

[0004]

The histamine-added γ-globulin which is an active ingredient of the pharmaceutical composition of the present invention is a complex of histamine and γ-globulin, and is prepared by appropriately mixing the γ-globulin component and the histamine component. can do. The human γ-globulin used in the present invention can be obtained from serum or placenta plasma or the like by a usual method, and as the histamine component, free histamine or its hydrochloride,
Pharmaceutically acceptable salts such as phosphate and picrate can be used. When producing the pharmaceutical composition of the present invention, for example, 1 to 200 mg, preferably 5 to 50 mg
g of γ-globulin and 0.01 to 2 μg, preferably 0.05 to 0.5 μg, of the histamine component are appropriately mixed.

[0005] Since the composition of the present invention is mainly used as an injection, it can be formulated as an isotonic solution using distilled water for injection or physiological saline. During the production, in addition to the γ-globulin component and the histamine component, additives such as a solubilizing agent, an isotonic agent, a stabilizer, a buffer, and a preservative can be appropriately added. For example, citric acid and benzoic acid Sodium, glycine, sodium sulfite, sodium bisulfite, sodium pyrosulfite, sodium thiosulfate, cysteine hydrochloride, phosphate, sodium ascorbate, sodium chloride, sodium hydrogen carbonate and the like can be used.

The pharmaceutical composition of the present invention may be prepared as an injection for use after dissolving it at the time of use. Each component may be mixed in a dry state, or the mixed solution may be filled in a vial or the like and then frozen. It can be obtained by drying. When producing a dry preparation for injection, excipients such as glucose, mannitol, and sorbitol may be added, if necessary, in addition to the above additives. As an example of the dry preparation for injection, for example, a histamine-added human immunoglobulin preparation used clinically can be mentioned.

Next, an example of a method for preparing histamine-added γ-globulin will be described. Since mice were used as experimental animals in the following pharmacological tests, mouse γ-globulin was used instead of human γ-globulin. That is, mouse γ-
Globulin and histamine dihydrochloride were dissolved in the following ratio, stirred at room temperature for 2 hours, freeze-dried, and dissolved by adding physiological saline at the time of use.

[Compound of the present invention] [Amount of mouse γ-globulin] [Amount of histamine dihydrochloride] HG50 5.3 mg 0.10 μg HG75 12.0 mg 0.15 μg HG90 28.8 mg 0.30 μg Prepared HG50, HG75 and HG90 Shows a significant effect in any of the following pharmacological tests, and shows the results obtained with HG75 as a representative example.

[0009]

Function I. Immunomodulatory action The immunomodulatory action was measured using trinitrophenyl (TNP) -specific antibody production and TNP-specific delayed-type hypersensitivity (DTH) reaction as indices.

(1) Preparation of trinitrophenyl-bound sheep erythrocyte (TNP-SRBC) Trinitrobenzenesulfonic acid (TNBS) is dissolved in phosphate buffered saline (40 mg / 7.0 ml, pH 7.2), and One ml of sheep red blood cell pellet was added dropwise with stirring. After being allowed to stand at room temperature for 20 minutes while stirring several times in the light-shielded state, it was washed three times with physiological saline. After centrifugation at 3000 rpm for 5 minutes, the mixture was adjusted to 5 × 10 ⁹ / ml with physiological saline.

(2) Production of TNP-Specific Antibodies 10 to ⁹ TNPs were produced in 6-8 week-old female BALB / c mice.
-SRBC was intraperitoneally administered, and anti-TNP antibody in the serum was changed to dinitrophenyl-bovine serum albumin (DNP-BS
It measured by the enzyme immunoassay (ELISA) using A). As a result, the anti-TNP-I peaked on the fourth to sixth days.
Strong antibody production of gM and anti-TNP-IgG was observed. In BALB / c nude mice lacking thymus, production of both types of antibodies was hardly observed.

(3) TNP-specific DTH reaction After sensitization with TNP-SRBC in the same manner as in (2) above,
On the day, 0.025 ml of 4.7 mg / ml TNBS was injected into the right footpad of the hind limb to elicit a TNP-specific DTH response. At 24 hours and 48 hours after the induction, the thickness of both footpads was measured with a dial gauge, and the difference between left and right swelling was expressed as the intensity of the DTH reaction.
As a result, a clear DTH reaction was observed 24 hours after the induction, but no DTH reaction was observed in BALB / c nude mice. This DTH reaction measurement system can be tested using the same mouse as the antibody production system of the above (2).

(4) Measurement of Action of Test Drug Using the test system described above, the compound of the present invention, histamine-added mouse γ-globulin (150 mg / kg / day), cyclosporin A
(100mg / kg / day), cyclophosphamide (100mg / kg / da)
y), prednisolone (0.5 mg / kg / day) and levamisole (5 mg / kg / day), anti-TNP antibody production and TNP by subcutaneous administration for 4 days from the day of TNP-SRBC sensitization
The effect on specific DTH reactions was examined.

The results of the anti-TNP antibody production system are shown in FIG.
Table 1 shows the results of the P-specific DTH reaction system. In the following main test results, a significant difference between the average value and the control was determined using Student's t-test, and marked with *. (*: P <0.00
1]

[0015]

[Table 1]

II. Eosinophilia inhibitory effect (1) Ragweed pollen antigen-induced eosinophilia model 6-8 week old female BALB / c mice were treated with saline
Ragweed pollen extract diluted 1000-fold was sensitized by subcutaneous injection of 0.1 ml on the first day and the first day, and further 0.2 ml on the sixth, eighth and fourteenth days. Ragweed antigen diluted 1000 times on day 20.
The reaction was induced by injecting 2 ml into the mouse intraperitoneally. 24 after trigger
At time, peritoneal exudate cells were collected and stained with Giemsa to count the total number of cells, eosinophils, neutrophils, and mononuclear cells. As a result, the number of eosinophils peaked at 24 hours after induction.
In the BALB / c nude mice lacking T cells, infiltration into the peritoneal cavity of both eosinophils and neutrophils was not observed at all.

(2) Measurement of Effect of Test Drug Using the above-described eosinophilia model, the compound of the present invention, histamine-added mouse γ-globulin (3 mg / mouse / day), was subcutaneously twice a week until the day of induction for 3 weeks. After administration, the effect on eosinophilia was examined. Histamine (dihydrochloride), which is a component of histamine-added mouse γ-globulin, and mouse γ
The test was also performed by administering a considerable amount of each of -globulin alone. Furthermore, cyclosporin A (100 mg / kg / day), which is known as an immunosuppressant, was subcutaneously administered for 3 days from 2 days before induction to the day before the comparison.

Table 2 shows an example of the result.

[Table 2]

(3) Measurement of the Effect of the Test Drug Using a Model of Eosinophilia Induced by Platelet Activating Factor (PAF) PAF (5 μm) was administered to 6- to 8-week-old female BALB / c mice.
g / 200 μl) was injected intraperitoneally into mice to elicit a response.
Twenty-four hours after the induction, peritoneal exudate cells were collected and the number of eosinophils was counted. Using the eosinophilia model, the inhibitory effect of the compound of the present invention, histamine-added mouse γ-globulin, on eosinophilia was examined in the same manner as described above.

Table 3 shows an example of the results.

[Table 3]

[0021]

As is apparent from the results shown in FIG. 1, the compound of the present invention, histamine-added γ-globulin, has a remarkable potentiating effect on the production of IgG and IgM antibodies. However, the immunosuppressants cyclosporin A and cyclophosphamide significantly suppressed the production of both antibodies. On the other hand, levamisole, which is said to have an immunomodulatory effect with the corticosteroid prednisolone, did not significantly affect antibody production at this dose. On the other hand, as shown in Table 1, the compound of the present invention, histamine-added γ-globulin, showed a significant inhibitory effect on delayed type hypersensitivity (DTH) reaction, and similarly, cyclosporin A and cyclophosphamide showed similar effects. A clear inhibitory effect was observed. Prednisolone also showed a weak inhibitory effect, but levamisole did not show any significant effect.

As described above, the conventional immunosuppressants cyclosporin A and cyclophosphamide markedly suppressed both immune responses of IgG and IgM antibody production and DTH reaction, but the compound of the present invention, histamine-added γ-globulin, It showed an enhancing effect on antibody production and an inhibitory effect on DTH reaction. Thus, it was revealed that histamine-added γ-globulin has an immunomodulatory effect that is clearly different from conventional immunosuppressants.

Further, as shown in Table 2, the compound of the present invention, histamine-added γ-globulin, markedly suppressed eosinophil infiltration into the peritoneal cavity in a model of eosinophilia induced by ragweed pollen antigen. Further, as is clear from the results in Table 3, histamine-added γ-globulin has a clear inhibitory effect on eosinophilia induced by PAF administration but not by antigen induction, similarly to the immunosuppressant cyclosporin A. Indicated. This eosinophilia-suppressing effect is not observed in the histamine and γ-globulin components alone, which are the components of histamine-added γ-globulin, and it is apparent that the effect is specific to the compound of the present invention. In the same administration period as cyclosporin A (2 days before induction to 3 days from the day of induction),
A significant inhibitory effect was observed in the above antigen-induced and PAF-induced eosinophilia models.

As is clear from the above pharmacological test results,
Since the pharmaceutical composition of the present invention has a specific immunomodulatory action distinctly different from conventional immunosuppressants, diseases in which the immune system has become abnormal, such as rheumatoid arthritis, systemic erythematosus, multiple sclerosis, etc. It is useful as a drug for treating autoimmune diseases and various immunodeficiency syndromes.

The pharmaceutical composition of the present invention has an inhibitory effect on eosinophilia. Eosinophils are known as effector cells that gather at sites stimulated by inflammation and cause inflammatory symptoms. Therefore, a drug that suppresses eosinophilia can be used as a drug that suppresses inflammation. The pharmaceutical composition of the present invention containing histamine-added γ-globulin as an active ingredient, in addition to the eosinophilia-suppressing action,
Since it also has an action of suppressing swelling in the DTH reaction, it is very useful as an excellent anti-inflammatory agent.

[0026]

The following is an example of the formulation of the pharmaceutical composition of the present invention, but the present invention is not limited thereto. The dose is preferably set as appropriate according to the type and severity of the disease, the age and sex of the patient, the administration period, and the like.

[Table 4]

[Brief description of the drawings]

FIG. 1 is a graph showing the enhancing effect of the pharmaceutical composition of the present invention on anti-TNP antibody production.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 37/00 A61P 37/02 37/02 C07D 233/64 105 // C07D 233/64 105 A61K 37/04 F-term (reference) 4C084 AA02 BA01 CA36 DA38 MA02 NA14 ZA02 ZA96 ZB05 ZB07 ZB11 4C086 AA01 AA02 BC38 MA02 NA14 ZA02 ZA96 ZB05 ZB07 ZB11

Claims (7)

[Claims] 1. An immunomodulator containing histamine-added γ-globulin as an active ingredient. 2. The therapeutic agent according to claim 1, which is an autoimmune disease therapeutic agent. 3. The therapeutic agent according to claim 2, which is a multiple sclerosis therapeutic agent. 4. The therapeutic agent according to claim 2, which is a therapeutic agent for systemic lupus erythematosus. 5. The method according to claim 2, which is a therapeutic agent for rheumatoid arthritis.
The therapeutic agent as described above. 6. The therapeutic agent according to claim 1, which is an immunodeficiency syndrome therapeutic agent. 7. An anti-inflammatory agent containing histamine-added γ-globulin as an active ingredient.