JPH11246432A - Immunosuppressants - Google Patents

Abstract

(57) [Summary] (Modified) [PROBLEMS] To provide a drug having an immunosuppressive action. SOLUTION: It contains a partial peptide represented by the amino acid sequence of SEQ ID NO: 2 or 3 as an active ingredient. SEQ ID NO: 2 Asp His Gln Tyr Tyr Asn A
sp Phe SEQ ID NO: 3 Asp Pro Ser Tyr Val Asn V
al Gln Asn

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an immunosuppressant comprising a certain peptide as an active ingredient, and more particularly, to an immunosuppressant.
hc protein (Shc protein) CH region (α1 homologou
The present invention relates to an immunosuppressant comprising, as an active ingredient, a partial peptide containing a tyrosine residue in s collagen region).

[0002]

BACKGROUND OF THE INVENTION It is presently believed that many intractable diseases involve an abnormal immune response. For example, systemic lupus erythematosus (SLE), a representative example of intractable autoimmune diseases
Induces various serious organ injuries including lupus nephritis by immune responses to self-antigens such as antinuclear antibody production (Kelly, WN et al., Textbook of Rheumatolog
y II third edition 1077-1129 1986). In addition, allergic diseases such as atopic dermatitis are also immune diseases caused by an excessive immune response.

[0003] In the treatment of such autoimmune diseases, administration of drugs that suppress the entire immune response is mainly performed. Specific examples include steroids and immunosuppressants such as cyclosporin A, but these drugs have significant side effects,
Also, the degree of completion is not high, such as not being tissue-specific. Currently, immunosuppressants used for medical purposes include corticosteroids, antimetabolites, alkylating agents, alkaloids,
Antibiotics, anti-lymphocyte antibodies and the like are known. Specific examples include immunosuppressants such as steroids and cyclosporin A. However, such drugs are not perfect because they have large side effects and are not tissue-specific. Therefore, effectiveness,
There is a need for the development of immunosuppressants that are more excellent in terms of durability and side effects.

[0004] T cells, which are normally in a quiescent state, are quickly activated in response to the invasion of a foreign antigen, proliferate themselves, and elicit various immune responses through production of cytokines. The detailed mechanism of this T cell activation is currently being elucidated. T
The outline of the mechanism of cell activation is as follows.
(T cell receptor: T cell antigen receptor) complex is phosphorylated, and a 70 kDa tyrosine kinase called ZAP-70 (zeta-associated protein 70), which is specifically expressed in T cells, binds to the complex. ZAP-70, which plays a central role in T cell activation, is further phosphorylated and activated by Lck. As a further step, we have found that a protein called Shc is phosphorylated by ZAP-70 and that this phosphorylation is essential for T cell activation (Iwashi
maet al., posting). Thus, since T cells play a central role in regulating immune responses, TAP-
Phosphorylation of Shc by 70 is extremely important in vivo.

[0005] The entire amino acid sequence of this Shc is Giul
(Cell, vol. 70, 93-105, Jul.
y10, 1992), and Millia et al. also reported that the PTB domain (aminoterminal phosphotyr
(Oncogene 1996, 13, 767-). It has been reported that a sine binding domain) is involved in phosphorylation by ZAP-70, and that T cell activation is suppressed when a PTB domain is expressed in T cells. 775).

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies to develop more excellent immunosuppressants, and as a result, Sh
When a peptide containing a phosphorylation site by ZAP-70 of c protein, specifically, a partial peptide containing a tyrosine residue which is a phosphorylation site in the CH region of Shc protein is expressed in an immunocompetent cell (T cell). It has been found that the effect of suppressing the activation of the immunocompetent cells is high. The present invention has been accomplished based on these findings.

That is, the present invention is an immunosuppressant comprising a partial peptide containing a tyrosine residue in the CH region of the Shc protein, a salt thereof or a hydrate thereof as an active ingredient. Also,
According to a preferred embodiment of the present invention, the above immunosuppressant wherein the partial peptide is the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing; the tyrosine residue is the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing The above-mentioned immunosuppressive agent, which is one or more tyrosine residues selected from the 7th, 8th, and 85th tyrosine; a partial peptide containing the tyrosine residue, the tyrosine residue and the preceding and / or following tyrosine residue; The above immunosuppressive agent, which is a peptide containing a plurality of amino acid residues; the above immunosuppressant, wherein the partial peptide containing a tyrosine residue is a peptide having 5 to 10 amino acid residues; the portion containing a tyrosine residue The above-mentioned immunosuppressant wherein the peptide is represented by the amino acid sequence of SEQ ID NO: 2 or 3 in the sequence listing; a partial peptide having the amino acid sequence represented by SEQ ID NO: 2 or 3 in the sequence listing; Or immunosuppressive agent comprising as an active ingredient mixtures of their hydrates are provided.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail. In the immunosuppressive agent of the present invention, the CH region of the Shc protein used as the active ingredient is not particularly limited, and specifically, Giuliana et al., Cell,
70, July 10, Issue 93-105 (1992)
233 to 377 in the amino acid sequence of the Shc protein described in
And a CH region represented by SEQ ID NO: 1 in the sequence listing having a primary structure of 145 residues represented by the amino acid sequence at the 番 目 th position. When the amino acid sequence described in SEQ ID NO: 1 in the sequence listing is used as the primary structure of the CH region of the Shc protein, the 7th, 8th, or 85th tyrosine of the same sequence is phosphorylated in vivo.

In the present invention, the seventh in the above sequence,
It is preferable to use, as an active ingredient, a partial peptide of the CH region containing one or more tyrosine residues (phosphorylation sites) selected from the eighth and 85th tyrosine. In the present invention,
The partial peptide in the CH region preferably used is preferably a peptide containing a tyrosine residue as the phosphorylation site and a plurality of amino acid residues before and / or after the tyrosine residue. In particular, a peptide containing 1 to 9 amino acid residues, more preferably 3 to 5 amino acid residues before or after the tyrosine residue at the phosphorylation site, or more preferably 5 to 10 amino acid residues Peptides are preferred. Further, among these partial peptides, a peptide having an amino acid sequence represented by SEQ ID NO: 2 or 3 in the sequence listing is most preferable,
A mixture of peptides having the amino acid sequences represented by SEQ ID NOs: 2 and 3 is more preferred. Furthermore, the partial peptide in the CH region used in the present invention may contain a tyrosine residue, and may be one in which some amino acids have been removed, substituted, modified or added.

[0010] The partial peptide containing a tyrosine residue in the CH region of the Shc protein, which is an active ingredient of the immunosuppressant of the present invention, can be obtained by a commonly used peptide synthesis method (Current Prot
ocols in Immunology, John, E., Coligan et al., publish
ed by Current Protocols) or a method equivalent thereto, or genetic recombination (Sambrook, J., Fritsch, EFa)
nd Maniatis, T., Molecular Cloning A laboratory ma
nual second edition, Cold Spring Harbor Laboratory
Press 1989).

As the immunosuppressant of the present invention, the above-mentioned peptide in free form, a pharmacologically acceptable salt thereof, or any hydrate thereof can be used. As pharmacologically acceptable salts, for example, hydrochloride, hydrobromide,
Mineral salts such as hydroiodide, sulfate, nitrate and phosphate, or acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malic acid Organic salts such as salts, mandelates, methanesulfonates, p-toluenesulfonates, and 10-camphorsulfonates, and sodium, potassium, lithium, magnesium, calcium, and ammonium salts. Can be mentioned.

As the immunosuppressant of the present invention, the above-mentioned peptide itself which is an active ingredient may be used, but a pharmaceutical composition (formulation) containing the above-mentioned active ingredient is produced using a general-purpose pharmaceutical additive. It is preferable to use them. Particularly preferred formulations include lotions, aerosols (sprays), liquid preparations, external preparations such as ointments, injections, etc., but are not limited thereto.

These preparations can be prepared by a commonly used method known per se. In formulating, if necessary, commonly used additives such as stabilizers, excipients, solubilizers, antioxidants, buffers, soothing agents, tonicity agents and the like may be used. The immunosuppressant of the present invention can be administered by an appropriate route depending on the form of the preparation. The dose of the immunosuppressant of the present invention may be appropriately determined depending on the age, health condition, body weight, severity of disease, kind and frequency of concurrent treatment / treatment, nature of desired effect, and the like of the patient. Generally, the dosage per adult is 1 to 100 mg, preferably 5 to 100 mg, as the amount of the active ingredient.
It may be administered once or several times a day as 50 mg.

[0014]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the scope of the present invention. Example 1 Preparation of Peptide Peptide Synthesizer (Biosyn 200 from Applied Biosystems)
0) and according to the standard protocol of the instrument based on 9-fluorenylmethoxycarbonyl (Fmoc) -peptide solid phase synthesis, the amino acid sequence of the CH region of Shc (SEQ ID NO: 1) A peptide having the amino acid sequence shown in No. 3 was synthesized. In the synthesis, His, Asn, and Gln are trityl groups; Tyr is a tertiary butyl group; Asp and Ser are Fmoc amino acid derivatives each having a tertiary butyroxycarbonyl group as a side chain protecting group, and other Fmoc amino acid derivatives All used those which did not contain a side chain protecting group.

These Fmoc amino acid derivatives are dissolved in N-methylpyrrolidone (NMP) and p-hydroxymethylphenoxyacetamidomethyl resin (amino group 0.
The peptide was synthesized using 4 equivalents of the Fmoc amino acid derivative with respect to 1 mmol). The coupling reaction between the Fmoc amino acid derivative and the resin is based on benzotriazole-
1-yl-oxy-tris-pyrrolidino-phosphonium-hexafluorophosphate (PyBOP) 1
Equivalents and N-hydroxy-benzotriazole (HoB
t) Performed using 1 equivalent and N, N-diisopropylethylamine (DIEA).

After completion of the synthesis, the protecting group and peptide are removed with a solvent consisting of 87.5% trifluoroacetic acid (TFA), 5.0% phenol, 2.5% triisopropylsilane, and 5.0% water. Separation was performed, and the peptide was purified using a Poros R2H column (manufactured by Perseptive biosystems). Example 2 Preparation of Peptide-Introduced Cells In this example, the peptide prepared in Example 1 was introduced into T cells, and T cell activation inhibitory activity was examined.

As a T cell line, a human cultured cell line J
urkat (ATCC TIB 152; J. Immunol. 133: 123-128,
1985). The medium was RPMI1640 medium (Gi
bcoBRL), 10% fetal bovine serum immobilized at 56 ° C. for 30 minutes, 50 μM 2-mercaptoethanol,
And one each of penicillin and streptomycin.
The one added so as to be 00 μg / mg was used.

The peptide (50 μg) prepared in Example 1 and NF-AT were added to Jurkat cells (5 × 10 ⁶ ).
A reporter plasmid (NF-AT-luc; J. Biol. Chem. 268;
917-2923, 1993) was introduced by electroporation (Cell porator, Gibco BRL) at 37 ° C.
The cells were cultured in the presence of 5% CO ₂ to obtain target peptide-introduced cells. Example 3 Inhibitory Effect of T Cell Activation After culturing the cells prepared in Example 2 for 1 day, anti-TCRβ
A chain antibody (prepared by the method described in J. Immunol. 134: 663-665, 1985) was added at a dilution of 1: 1000 to stimulate the cells, and the cells were further cultured for 10 hours.

After completion of the culture, the cells were collected, and the luciferase activity was measured using a luminometer (Lumat LB9501, Berthold), and used as an index of T cell activation. The activity was measured using Picka Gene Kit (Wako Pure Chemical Industries, Ltd.). As a result, as shown in FIG. 1, the cells into which the peptide prepared in Example 1 was introduced had significantly suppressed T cell activation as compared to the control (None) into which the peptide had not been introduced.

[0020]

Industrial Applicability The immunosuppressive agent of the present invention has an inhibitory effect on T cell activation and is useful for the prevention and / or treatment of diseases caused by abnormal lymphocyte activation, especially autoimmune diseases. It is.

[0021]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 145 Sequence type: amino acid Topology: Linear Sequence type: Peptide Glu Pro Pro Asp His Gln Tyr Tyr Asn Asp Phe Pro Gly Lys Glu Pro 1 5 10 15 Pro Leu Gly Gly Val Val Asp Met Arg Leu Arg Glu Gly Ala Ala Pro 20 25 30 Gly Ala Ala Arg Pro Thr Ala Pro Asn Ala Gln Thr Pro Ser His Leu 35 40 45 Gly Ala Thr Leu Pro Val Gly Gln Pro Val Gly Gly Asp Pro Glu Val 50 55 60 Arg Lys Gln Met Pro Pro Pro Pro Pro Cys Pro Gly Arg Glu Leu Phe 65 70 75 80 Asp Asp Pro Ser Tyr Val Asn Val Gln Asn Leu Asp Lys Ala Arg Gln 85 90 95 Ala Val Gly Gly Ala Gly Pro Pro Asn Pro Ala Ile Asn Gly Ser Ala 100 105 110 Pro Arg Asp Leu Phe Asp Met Lys Pro Phe Glu Asp Ala Leu Arg Val 115 120 125 Pro Pro Pro Gln Ser Val Ser Met Ala Glu Gln Leu Arg Gly Glu 130 135 140 Pro 145 Sequence No .: 2 Sequence length: 8 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Asp His Gln Tyr Tyr Asn Asp Phe 15 SEQ ID NO: 3 Sequence Is: 9 sequence type: amino acid Topology: linear sequence type: peptide Asp Pro Ser Tyr Val Asn Val Gln Asn 1 5

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of a test of immunosuppression when a partial peptide containing a tyrosine residue in the CH region of the Shc protein was introduced into T cells. In the figure, # 239 on the horizontal axis is SEQ ID NO: 2.
# 317 represents the peptide of SEQ ID NO: 3,
Unstim. Shows the results when the cells were not stimulated with the anti-TCRβ chain antibody, and anti-TCR shows the results when the cells were stimulated with the anti-TCRβ chain antibody.

Claims (7)

[Claims] 1. An immunosuppressant comprising a partial peptide containing a tyrosine residue in the CH region of the Shc protein, a salt thereof or a hydrate thereof as an active ingredient. 2. The immunosuppressant according to claim 1, wherein the partial peptide is represented by the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. 3. The tyrosine residue according to claim 1, wherein the tyrosine residue is at least one tyrosine residue selected from the 7th, 8th, and 85th tyrosine in the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. Immunosuppressants. 4. The immunosuppression according to claim 1, wherein the partial peptide containing a tyrosine residue is a peptide containing the tyrosine residue and a plurality of amino acid residues before and / or after the tyrosine residue. Agent. 5. The partial peptide containing a tyrosine residue is 5
The immunosuppressant according to any one of claims 1 to 4, which is a peptide having from 10 to 10 amino acid residues. 6. The immunosuppressive agent according to claim 1, wherein the partial peptide containing a tyrosine residue is represented by the amino acid sequence of SEQ ID NO: 2 or 3 in the sequence listing. 7. An immunosuppressant comprising, as an active ingredient, a partial peptide having the amino acid sequence represented by SEQ ID NOs: 2 and 3 in the sequence listing, a salt thereof, or a mixture of hydrates thereof.