JPH0489433A - Immunomodulator - Google Patents

Abstract

PURPOSE:To obtain an immunomodulator useful for treating autoimmune disease, having high medicinal effect, low side effects, readily usable, comprising a specific pentapeptide or a salt thereof as an active ingredient. CONSTITUTION:An immunomodulator comprising a pentapeptide shown by the formula (Asp is L-aspartic acid residue; Ser is L-serine residue; gly is glycine residue; Lysine residue) or a pharceutically permissible salt thereof useful as an antiallergic agent as active ingredient. A dose of the peptide is 0.01-10mg/kg/ day in the case of oral administration and 10-1,000mug/kg/day in the case of parenteral administration. The peptide supresses or enhance activation reaction of lymphocyte irritated with mitogen and raises productivity of liquid factor of IL-6 and TNF, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an immunomodulator useful for treating autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus.

[Prior Art] Conventionally, chemically synthesized drugs such as steroid hormones, gold preparations, D-penicillamine, and levamisole have been mainly used as therapeutic agents for so-called autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. Recently, interferon and interleukin (IL-1, I
Protein-based biological agents such as L-2, IL-3, etc.) have attracted attention, and various basic and applied studies are being conducted.

[Problem to be solved by the invention]

However, the above chemically synthesized drugs sometimes cause serious side effects, such as adrenal cortical insufficiency, infection, kidney damage, hematopoietic disorder, or gastrointestinal disorder, so extreme caution must be taken when using them. Protein-based biological agents such as interferon and interleukin are not always satisfactory in terms of efficacy.

The present invention provides an immunomodulator that is highly effective, has few side effects, and is easy to use.

[Means to solve the problem]

The present inventors have synthesized various peptides derived from the Fc region of IgE or peptides similar thereto, and have been intensively investigating their various pharmacological activities including antiallergic activity. The present invention was completed based on the discovery that some of them exhibit immunomodulatory effects. Here, the term "immunomodulatory effect" refers to an effect that acts to enhance immune function in a state of decreased immune function and to act to suppress a state of immune function that is enhanced, and a drug having this effect is referred to as an immunomodulatory agent.

That is, the present invention has the following formula [I] H-Asp-Ser-Asp-Gly-Lys-OH[
I ] (where ASp is L-aspartic acid residue, S
er is an L-serine residue, Gly is a glycine residue, Lys
represents an L-lysine residue) or a pharmaceutically acceptable salt thereof as an active ingredient.

The above H-Asp-Ser-Asp-Gly-Lys-
OH is a peptide discovered by the present inventors to be useful as an anti-allergic agent (Japanese Patent Application Laid-Open No. 1-3
No. 16398).

The above pentapeptide can be produced by a conventional solid phase method as described in JP-A-1-316398, or by Boc-Gly as described in JP-A-1-258366. -OH (However, Bo
Glycine derivatives represented by H-Lys(Z)-OBzl (where Z is a benzyloxycarbonyl group and Bzl is a benzyl group) and monolysine derivatives represented by The derivative was subjected to dehydration condensation to give Boc-Gly-Lys (Z)-0Bzl, the BoC group was removed with acid, and Boc-Asp (OBz
l) A mono-aspartic acid derivative represented by -0H is added and dehydrated to form Boc-Asp(OBzl)-Gly.
-Lys(Z)-0Bzl, remove the BoC group with acid,
A monoserine derivative represented by Boc-Ser-OH was added to this, and the mixture was dehydrated and condensed to form BoC-8erAsp (OB
zl)-Gly-Lys(Z)-0Bzl and B with acid.
It can also be obtained by removing the oC group, adding a monoaspartic acid derivative represented by Z-Asp(OBzl)-OH, dehydration condensation, and then catalytic reduction.

Pharmaceutically acceptable salts of the pentapeptide represented by H-Asp-Ser-Asp-Gly-Lys-OH include inorganic acid salts such as hydrochloride, sulfate, and phosphate; acetate;
organic acid salts such as citrate, fumarate, tartrate, lactate; alkali metal salts such as sodium salt and potassium salt;
Examples include alkaline earth metal salts such as calcium salts and magnesium salts, ammonium salts, and the like.

The above pharmaceutically acceptable salts can be prepared by removing the protecting group in the final step of synthesis and then adding an acid such as hydrochloric acid or acetic acid, or adding a base such as sodium hydroxide or potassium hydroxide to form the corresponding salt. Alternatively, after isolating the pentapeptide represented by formula CI), an acid or base can be added to form a salt in the same manner as above.

The immunomodulator of the present invention can be administered orally or parenterally as appropriate. Typical methods of administration include oral, rectal, percutaneous, subcutaneous, intravenous, intramuscular, inhalation, or intranasal routes.

In these administration methods, the immunomodulators of the invention can be administered in the form of various pharmaceutical formulations. The forms of these pharmaceutical preparations include tablets, hard capsules, soft capsules, granules, powders, troches, suppositories, syrups, creams, ointments, poultices, injections, suspensions, There are inhalers, aerosols, etc. It can also be made into double-layer tablets, multi-layer tablets, etc. together with other immunomodulators and other medicines.

Furthermore, in the case of tablets, a regular coating is applied as necessary.
For example, sugar-coated tablets or enteric-coated tablets can be used.

When preparing solid preparations such as tablets, granules, and powders, known additives such as lactose, sucrose, glucose, crystalline cellulose, cornstarch, calcium phosphate, sorbitol, glycine, carboxymethyl cellulose, and hydroxypropyl cellulose are used in the preparation. , gum arabic,
Polyvinylpyrrolidone, polyethylene glycol, magnesium stearate, talc, etc. can be added.

For semi-solid formulations, materials such as vegetable waxes, microcrystalline waxes, fats such as tarolanoline can be added.

When preparing a liquid formulation, additives such as sodium chloride, sorbitol, glycerin, olive oil, almond oil, propylene glycol, ethylene gelicol, and ethyl alcohol can be added.

The dosage of the peptide represented by formula CI) depends on the patient's age,
The dosage can be adjusted as appropriate depending on body weight, symptoms, etc., but the dosage for oral administration is 0.01 to 10 mg per day.
/kg, the amount for parenteral administration is 10 to 1 kg per day.
000μg/kg.

[Example] In the following example, it will be explained that the peptide represented by formula [I] has an immunomodulatory effect and can be used for treatment as an immunomodulator.

(Example 1) Expression on the blastogenesis of lymphocytes stimulated with various lectins [
Effect of the peptide represented by peptide I] Ficoll-Hypacu
Mononuclear cells were collected by specific gravity centrifugation (1-Hypaque), and fetal bovine serum (abbreviated as FBS, manufactured by Dainippon Pharmaceutical Co., Ltd.) was added to
This was suspended in RPMI-1640 culture solution (manufactured by Gibco) to which % by volume was added. Concentration of cell number: lx106 cells/m
100 μl of this was dispensed into each well of a 96-well microplate (manufactured by Falcon).

Then PHA (Phytohemagglutin)
in) 1 ug/ml, Con-A (Con
canavalin A) 10 μg/m 1 or PWM (Pokeweed mitogen)
After adding 1511 g/ml of each lectin (all manufactured by Dai-Kyakuhin) and a predetermined concentration of peptide [I], and adding RPMI-1640 medium supplemented with 10% FBS to make the final volume 200 μl, The cells were cultured at 37°C for 72 hours in a 5% CO2 incubator. Culture completed 2
Add 0.5 μCi of 3H-thymidine 4 hours before cell
Cells were harvested using a harvester (manufactured by Bio-Lab), and the amount of 3H-thymidine uptake was measured using a liquid scintillation counter. Table 1 shows the measurement results, and the numbers in the table show the average counts (cpm) and standard errors.

From the results in Table 1, the peptide of formula [I] has little effect on the amount of 3H-thymidine uptake by lymphocytes when mitogen is not added, but each lectin, especially Co
It can be seen that the amount of 3H-thymidine uptake by lymphocytes stimulated with n-A is strongly suppressed.

Table 1 (Continued) (Example 2) Effect of peptide represented by formula CI on humoral factor production ability Mononuclear cells separated by Ficoll-Hypaque density centrifugation from healthy individuals whose blood was collected in the presence of heparin were ,FBSIO
% by volume was suspended in RPMI-1640 culture medium. Insert this into a culture tube (Falcon 2054 tube)
The peptide represented by formula [1] was added to the solution at 106 Ix cells/ml, and cultured in an incubator for one week.

After the culture was completed, the cells were centrifuged (1.50 rpm, 10 minutes), the supernatant was collected, and the humoral factors in the culture supernatant were measured.

interleukin-1 (IL-1) and tumor necrosis factor (
TNF) was measured using radioimmunoassay kits from Amajam and Medginix, respectively. Interleukin 6 (IL-6) was measured by enzyme-linked immunosorbent assay (ELISA) using anti-IL-6 antibody (manufactured by Dienzyme). The measurement results are shown in Table 2. Note that the numerical values represent the standard error above the mean.

Table 2: Lymphocyte production capacity for humoral factors From the results in Table 2, it can be seen that the peptide of formula (I) strongly promotes the production of IL-6 and TNF.

[Effect of the invention] The peptide represented by formula CI) suppresses or enhances the activation reaction of lymphocytes stimulated with mitogen, and also
Enhances production of humoral factors such as -6 and TNF. According to the present invention, it was possible to provide a drug that is effective as an immunomodulator, has few side effects, and is easy to use.

Claims (1)

[Claims] 1. The following formula [I] H-Asp-Ser-Asp-Gly-Lys-OH [
I] (where Asp is L-aspartic acid residue, S
er is an L-serine residue, Gly is a glycine residue, Lys
is an L-lysine residue) or a pharmaceutically acceptable salt thereof as an active ingredient.