JPH06336428A - Immunosuppressant - Google Patents

Abstract

PURPOSE:To obtain an immunosuppressant containing an epoxysuccinic acid derivative. CONSTITUTION:It has been found that cathepsin B is involved in antigen processing, and the immunosuppressant containing an epoxysuccinic acid derivative having the enzyme inhibitory activity has been produced. This agent can suppress induction of immunity by such inhibitory activity, and so can be used as a medicine for various kinds of autoimmune diseases.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to an immunosuppressant. More specifically, it relates to an autoimmune disease therapeutic agent that specifically inhibits cathepsin, which is an antigen processing enzyme.

2. Description of the Related Art The mechanism of antigen processing or presentation in vivo is generally considered as follows. First, the antigen is taken up by antigen-presenting cells such as macrophages, dendritic cells, and B cells by endocytosis, and digested by the endosome-degrading enzyme in the acidic fraction to form an antigenic peptide consisting of 10 to 20 amino acids. Is decomposed (Processing). The antigenic peptide is then cytosolic in the class II major histocompatibility complex.
y complex (MHC) molecule. Finally, the complex is expressed on the antigen presenting cell membrane. CD4-positive helper T cells recognize an antigen bound to a class II MHC molecule, are activated, and proliferate, thereby stimulating B cells to divide and proliferate, differentiate into plasma cells and secrete antibodies [Akihiko Yano, Clinical Immunity, 2
2,511-522 (1990); Aizawa Shiro, Clinical Immunity, 22,523-533 (1990); M. Gray, A. Sette, S. Booth, Scientific
American Japanese Version, 20, 7-17 (199
0)]. The T cell antigen receptor (TCR-α, β chain) that recognizes the peptide-MHC complex and the gene encoding the same have been elucidated, and in order for T cells to react, two types of antigens and self MHC products are required. Must be recognized by the double-stranded TCR and its antigen specificity and M
It has been found that HC-restriction is determined. In order for many antigenic peptides to bind to class II MHC molecules, it is believed that the antigenic peptides adopt a regular ordered structure, but it is not clear what is defined by them. Also, the proteases involved in the processing of immunological fragments of the antigen have not been revealed to date.

As described above, the antigen is processed in the endosome of the acidic fraction and decomposed into about 10 to 20 amino acids, and the resulting short peptide binds to the class II MHC molecule and becomes a cell. Presented on the surface. To date, the identity of the enzyme involved in the processing of this antigen is unknown, and by clarifying this stage, we elucidated the interaction between the MHC molecule and the antigen,
It is desired to develop a medicine that suppresses this. These clarifications open the way to the treatment of autoimmune diseases in which the immune system responds to its own molecules. Specifically, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and insulin-dependent Diabetes (Type I diabetes), inflammatory bowel disease, systemic lupus erythematosus, glomerulonephritis, autoimmune hemolytic anemia, Hashimoto's disease, ulcerative colitis, primary biliary cirrhosis, idiopathic thrombocytopenic purpura, exchange It will lead to the development of remedies for Ophthalmitis, Pernicious Anemia, Sjogren's Syndrome, Goodpasture Syndrome, etc.

Based on such research background, the present inventors have found that if the production of a specific antibody against an antigen is suppressed by the administration of a highly specific protease inhibitor, the antigen processing is performed. As a result of intensive investigations, it was possible to identify a protease involved in Escherichia coli. As a result, a cysteine protease inhibitor, particularly an epoxysuccinic acid derivative and an anti-cathepsin B inhibitor which are cathepsin B inhibitors.
It was found that the antibody inhibits the presentation of the antigen and suppresses the production of the specific antibody. Furthermore, the primary structure and three-dimensional structure of cathepsin B and the primary structure of class II MHC molecule were compared and examined, and one of the active centers of cathepsin B, VN
_217-223 (-VANSWNT-) (SEQ ID NO: 1)
Is the binding domain of the class II MHC molecule β chain, VN
_57-63 (-VAESWNS-) (SEQ ID NO: 2) shows high homology, and among the highly homologous sequences, other cathepsins differ in that they are a lysine residue and a glycine residue, respectively. It was found that alanine residue and asparagine residue are the same amino acid between cathepsin B and class II MHC molecule, and the antigenic peptide processed by cathepsin B specifically binds to class II MHC molecule to present antigen. I made a prediction that this would occur [Fig. 1]. On the other hand, since the class II MHC β chain shows an affinity with the C-terminal domain sequence of the antigen peptide, it can be said from the above prediction that this affinity is defined by the substrate specificity of cathepsin B. Therefore, the present inventor has conducted extensive studies on various peptides produced by the degradation of cathepsin B, and found peptide sequences showing a common affinity for class II MHC molecules,
The present inventors have completed the present invention by clarifying that the antigenic presentation domain is composed of the sequence and a peptide added to the N-terminal side thereof. That is, the present invention provides (1) an immunosuppressive agent containing an epoxysuccinic acid derivative, (2) an immunosuppressive agent according to (1) above, which contains an epoxysuccinic acid derivative having a cathepsin B inhibitory action, and (3) autoimmunity. The immunosuppressive agent according to (1) above, which is a therapeutic agent for diseases. The epoxysuccinic acid derivative according to the present invention is a product obtained by modifying the carboxylic acid or a salt thereof, and has an action capable of inhibiting the activity of a protease (thiol protease) whose thiol group in the molecule is considered to be the active center. Say something. Examples of the carboxylic acid derivative of epoxysuccinic acid include mono- or diesters of trans epoxysuccinic acid, hydroxamic acid, mono- or diamide. The trans epoxy succinate ester has the formula ◎

[Chemical 1] [Wherein, R ¹ and R ² are the same or independent of each other,
A hydrogen atom, an optionally substituted alkyl or cyclic hydrocarbon group is shown. ] The compound shown by these or its salt is mentioned. Examples of the alkyl which may be substituted in the above include a linear or branched alkyl group having 1 to 5 carbon atoms which may be substituted with a cyclic hydrocarbon group which may have a substituent. Preferred are linear alkyl groups substituted with an optionally substituted cyclic hydrocarbon group. The cyclic hydrocarbon group which may be substituted is 1-3 lower (C _1-3 ) alkyl group, lower (C _1-3 ) alkoxy or 1 to 3 cyclic group which may be substituted with a halogen atom. An alicyclic or aromatic hydrocarbon group is preferable, and among them, a 1 to 3 ring alicyclic or aromatic hydrocarbon group is a cycloalkyl group, a cycloalkenyl group or a cycloalkane group or a monocyclic aromatic hydrocarbon group. Some are preferable, and especially those having 3 to 3 carbon atoms.
8 monocycloalkyl group, bicycloalkyl group having 6 to 8 carbon atoms, tricycloalkyl group having 8 to 11 carbon atoms, monocycloalkenyl group having 5 to 8 carbon atoms, 5 carbon atoms
It is preferably a bicycloalkenyl group having 8 to 8, a monocycloalkene group having 6 to 9 carbon atoms, a bicycloalkene group having 6 to 9 carbon atoms, or an aromatic hydrocarbon group having 5 to 7 carbon atoms. In the present invention, R ¹ or R in the above
² are the same or independent of each other, hydrogen, an _optionally substituted C _8-9 aralkyl group, an _optionally substituted C _3-6 monocycloalkyl-C _1-4 alkyl group, C
₁₀ tricycloalkyl-C _1-2 alkyl group, C
_6-9 monomethyl cycloalkene _{methyl, C 6-9} bicycloalkene _{methyl, C 4-8} mono cycloalkyl group, an optionally substituted _{C 7} bicycloalkyl _{group, C 10} tricycloalkyl _{group, C 5-8} Epoxysuccinic acid or a salt thereof, which is a monocycloalkenyl group or a C _5-8 bicycloalkenyl group, is particularly preferably used, and specific examples thereof include, for example, epoxysuccinic acid cyclopentyl ester potassium salt and epoxysuccinic acid cyclohexyl ester sodium salt. , Epoxy succinic acid pentamethyl ester potassium salt, Epoxy succinic acid cyclohexane methyl ester potassium salt, Epoxy succinic acid norbornyl ester potassium salt, Epoxy succinic acid-
2-adamantyl ester potassium salt, epoxysuccinic acid-4-cyclooctenyl ester potassium salt, epoxysuccinic acid-3-cyclohexanepropyl ester potassium salt, epoxysuccinic acid di-4-cyclooctene methyl ester epoxydi (trans-2- Chlor) cyclopentane methyl ester [JP-A-54-141735]
And so on. The trans epoxy succinamide has the formula ◎

[Chemical 2] [In the formula, R ³ is a hydrogen atom, a monocyclic cyclic hydrocarbon, or a monocyclic cyclic hydrocarbon group having 1 to 10 carbon atoms which may be substituted.
10 represents a hydrocarbon or hydrogen residue, X represents N or O, and Y represents an amino group which may be substituted]. Examples of the monocyclic cyclic hydrocarbon group here include a cycloalkyl group, a cycloalkenyl group, a cycloalkene group, a cycloalkane group and an aromatic hydrocarbon group, and further, these are a halogen atom of 1 to 3 and a lower (C _1-3 ) Those substituted with an alkyl or lower alkoxy group are also included. Of these, a cycloalkyl group having 5 to 6 carbon atoms, a cycloalkenyl group having 5 to 6 carbon atoms, a cycloalkane having 5 to 6 carbon atoms and an aromatic hydrocarbon group having 5 to 6 carbon atoms are preferable. The chain hydrocarbon group includes straight chain or branched alkyl, alkenyl or alkynyl, preferably C
Examples of the chain hydrocarbon substituted with a _1-5 alkyl group, a C _2-4 alkenyl group, a C _2-4 alkynyl group, and a monocyclic hydrocarbon include C _5-6 cycloalkyl, C
_1-3 alkyl group, C _5-6 cycloalkene, C _1-3
An alkyl group and a C _1-3 aralkyl group are preferable. The amino group which may be substituted is an amino acid having a bond to N or ◎

[Chemical 3] [In the formula, R ⁴ and R ⁵ may be the same or independently, and may be bonded via a hydrogen atom, a C _1-5 alkyl group, or a lower (C _1-3 ) alkyl, and 1 as a ring-constituting group. 1 or 2 cyclic ring group optionally having N, S, O,
N-carboxyamino C _4-5 alkyl, or R ⁴ and R ⁵ form a 5- or 6-membered heterocyclic group together with the nitrogen atom to which it is bonded]. Here, the amino acid means an amino acid which may have 1 or 2 peptide bonds, its ester, carboxamide and salt, and the amino acid residue is L-α-amino acid, especially natural L-α-amino acid. Are preferred, and specific examples thereof include leucine, isoleucine, proline, threonine, tyrosine, and arginine. Specific examples having two amino acid residues include prolylproline, threonylisoleucine, tyrosinylarginine, isoleucylproline, isoleucylarginine and the like. The above-mentioned alkyl group may be linear or branched. N, S, O as a ring-constituting group
The 1- or 2-cyclic cyclic group which may have is specifically a cycloalkyl group (preferably C _3-6 cycloalkyl), a cycloalkenyl group, a cycloalkynyl group, C
_3-6 cycloalkane, C _1-3 alkyl group, aryl group (preferably phenyl), aralkyl group (preferably benzyl group, phenethyl group, etc.), thienyl group, tenyl group, furyl group, furfuryl group, pyridinyl group, quinolyl And the like, and these cyclic groups include 1 to 3 methyl groups, trifluoromethyl groups, methoxy groups, halogen atoms, acetyl groups, hydroxyl groups, nitro groups, carboxy groups,
It may have a substituent such as a carboethoxy group. R
Heterocyclic ^{group 2} and R ³ form together with their binding to the nitrogen atom, N in addition to the nitrogen atom to which R ² and R ³ are bonded, S
Or it may have any one hetero atom of O, and specific examples thereof include a pyrrolidine group, a piperazino group,
Examples thereof include a morpholino group and a thiamorpholino group, and these heterocyclic groups may be substituted with a methyl group, a carboxy group, a carboalkali metaloxy group, a carbomethoxy group, a carbobutoxy group, a carbobenzoxy group or the like.
Specific examples of the epoxysuccinamide include, for example, ethyl N-phenyl-2,3-epoxysuccinamate, ethyl N-2′-pyridinyl-2,3-epoxysuccinamate, ethyl N-8′-quinolinyl. -2
3-epoxysuccinamate, ethyl N-benzyl-
2,3-epoxysuccinamate [Japanese Patent Publication No. 55-476
68], N- (DL-3-trans-carboxyoxirane-2-carbonyl) -L-leucyl-L-proline methyl ester, N- [N '-(DL-3-trans-
Carboxyoxirane-2-carbonyl) -L-leucyl] phenethylamine potassium salt [JP-A-55-115]
878], N- (L-3-trans-carboxyoxirane-2-carbonyl) -L-leucyl agmatine [Agricultural and Biological Chemistry (Agric. Biol. Chem) Vol. 42, pp. 523-528]. (1978)], N- (L-3-trans-carboxyoxirane-2-carbonyl) -L
-Tyrosyl-N-nitro-L-arginine benzyl ester [JP-A-2-304074], N- (L-3-trans-ethoxycarbonyloxirane-2-carbonyl) -L-prolyl-L-proline [JP-A-2- 304
085], N- (L-3-trans-ethoxycarbonyloxirane-2-carbonyl) -L-isoleucyl-
L-proline, N- (L-3-trans-carboxyoxirane-2-carbonyl) -L-isoleucyl-L-
Proline, N- (L-3-trans-ethoxycarbonyloxirane-2-carbonyl) -L-isoleucyl-
2-proline sodium salt, N- (L-3-trans-
Methoxycarbonyloxirane-2-carbonyl) -L
-Isoleucil-L-proline [JP-A-3-4247
8], N- (L-3-trans-n-propylcarbamoyloxirane-2-carbonyl) -L-isoleucyl-L-proline (CA-074), N- (L-3-trans-n-peptylcarbamoyloxirane -2-Carbonyl) -L-isoleucyl-L-proline [Patent Document 4]
-139182] and the like. Among these epoxysuccinic acid derivatives, an epoxysuccinic acid amino acid amide derivative having a highly specific inhibitory effect on cathepsin B, particularly an epoxysuccinic acid amino acid amide derivative having two amino acid residues or a salt thereof is preferable. Used. Further, isoleucine or proline is preferably used as the amino acid residue. The epoxysuccinic acid derivative of the present invention can be produced according to a known chemical synthesis method. For example, regarding the epoxysuccinic acid ester derivative, the method described in JP-A-54-141735 and JP-B-60-37105, and regarding the epoxysuccinamide derivative, the method described in JP-A-55-47668, The epoxysuccinic acid amino acid amide derivative should be produced according to the method described in JP-A-2-304074, JP-A-2-304075, JP-A-3-72478, and JP-A-4-139182. You can The immunosuppressive agent of the present invention comprises an effective amount of the above epoxysuccinic acid derivative alone or in combination with a physiologically acceptable carrier to prepare a solid preparation such as tablets, capsules, granules and powders; or syrups and injections. Can be administered orally or parenterally as a liquid preparation such as. As the pharmaceutically acceptable carrier, various organic or inorganic carrier substances conventionally used as formulation materials are used, and excipients, lubricants, binders, disintegrants in solid formulations; solvents and solubilizing agents in liquid formulations , A suspending agent, a tonicity agent, a buffering agent, a soothing agent, etc. If necessary, formulation additives such as antiseptics, antioxidants, coloring agents and sweeteners can also be used. Suitable examples of excipients include lactose, sucrose, D-mannitol, starch, crystalline cellulose, light anhydrous silicic acid and the like. Preferable examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like. Preferable examples of the binder include crystalline cellulose, sucrose, D-mannitol,
Examples thereof include dextrin, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone and the like. Preferable examples of the disintegrant include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, carboxymethyl starch sodium and the like. Preferable examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil and the like. Preferable examples of the solubilizing agent include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like. Suitable examples of the suspending agent include, for example, stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, surfactants such as glyceryl monostearate; for example, polyvinyl alcohol, Examples thereof include hydrophilic polymers such as polyvinylpyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose. Preferable examples of the isotonicity agent include sodium chloride, glycerin, D-mannitol and the like. Preferable examples of the buffer include buffer solutions of phosphate, acetate, carbonate, citrate and the like. Preferable examples of soothing agents include benzyl alcohol and the like. Preferable examples of preservatives include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like. Preferable examples of the antioxidant include sulfite, ascorbic acid and the like.

It has already been clarified that the epoxysuccinic acid derivative used in the present invention has a strong inhibitory effect on cathepsin B and low toxicity. Therefore, the immunosuppressive agent of the present invention is a mammal (eg, mouse, rat, rabbit, dog,
It can be safely used for treating various autoimmune diseases of cats, cattle, pigs, humans, etc.). When the preparation of the present invention is used as a therapeutic agent for autoimmune diseases, the dose may vary depending on the type and condition of disease, administration method, age of the administration subject, sex, etc., but usually 10 to 10 times as an epoxysuccinic acid derivative per day. 2000 mg can be administered in 1 to several divided doses. In the present specification and drawings, when an amino acid is represented by an abbreviation, IUPAC-IUB Commi
sion on Biochemical Nomen
It is based on the abbreviations based on the "culture" or the abbreviations commonly used in this field, and examples thereof are given below. When amino acids may have optical isomers, the L form is shown unless otherwise specified. Gly: Glycine (G) Ala: Alanine (A) Val: Valine (V) L'eu: Leucine (L) Ile: Isoleucine (I) Ser: Serine (S) Thr: Threonine (T) Cys: Cysteine (C) 1/2 Cys: Half cystine Met: Methionine (M) Glu: Glutamic acid (E) Asp: Aspartic acid (D) Lys: Lysine (K) Arg: Arginine (R) His: Histidine (H) Phe: Phenylalanine (F) ) Tyr: Tyrosine (Y) Trp: Tryptophan (W) Pro: Proline (P) Asn: Asparagine (N) Gln: Glutamine (Q) [Examples] Reference examples and examples will be described in more detail below. However, the present invention is not limited to these. Reference Example 1 Inhibition of In Vivo Antibody Production by Cathepsin B Inhibitor A single dose of a sufficient amount of antigen administered to an experimental animal resulted in about 1 post-immunization.
Specific antibody was induced from day 0, and the antibody titer was about 2
Reach the highest level in the third week. If protease inhibitors were administered prior to immunizing animals with antigen and antibody production was significantly suppressed and antibody response was delayed, the enzymes involved in processing could be inferred. Based on this idea, various proteases were investigated. As a result, cysteine protease inhibitors, especially cathepsin B inhibitors (eg CA-074 (N-
(L-3-trans-n-propylcarbamoyloxirane-2-carbonyl) -L-isoleucyl-L-proline: JP-A-4-139182, E-64 (N- (L-
3-trans-ethoxycarbonyloxirane-2-carbonyl) -L-isoleucine-methylbutyramide;
(Taisho Pharmaceutical Co., Ltd.)) was intraperitoneally administered, and production of antibodies against the inoculated antigen was inhibited. Furthermore, when the cathepsin B inhibitor was administered every day after inoculation, the antibody production was almost completely suppressed. These results suggest that cathepsin B is involved in antigen processing, and that cathepsin B inhibitors can regulate specific antibody production. Reference Example 2 In Vitro of Sensitized Splenocytes by Restimulation of Antigen
Effect of Peptase Inhibitors on Proliferation of Stroocytes (Sensitized Spleen Cells) of Animals Immunized with Antigen
When restimulated with the antigen in vitro, the cells show a marked proliferative response. In this experimental system, the effects of various protease inhibitors on the proliferative response were investigated. That is, i.
Upon restimulation with the antigen in vitro, various proteases were added to investigate the proliferative response. As a result, it was found that the addition of cysteine protease inhibitors, especially cathepsin B inhibitors, markedly inhibited the proliferative response of splenocytes. In addition, when a cysteine protease inhibitor, particularly a cathepsin B inhibitor was simultaneously administered during immunization of splenocytes, the proliferative response of sensitized splenocytes by restimulation with the antigen was significantly inhibited. Furthermore, when the inhibitor was added during restimulation of splenocytes immunized in the presence of cathepsin B, proliferation was further suppressed. These results suggest that cathepsin B is involved in antigen processing. Reference Example 3 i of sensitized splenocytes by restimulation with antigen peptide
Effect of Protease Inhibitors on n Vitro Proliferation If the protease is involved in the processing of the antigen, the antigenic peptide degraded by that protease should promote the proliferative response of sensitized splenocytes. As a result of examination with various proteases based on this idea, a synthetic peptide corresponding to a cathepsin B degradation product (active peptide)
Found that the proliferative response of sensitized splenocytes was stimulated, but that the synthetic peptides corresponding to other domains did not show such a proliferative response of splenocytes. Further, the proliferative response by this active peptide was not inhibited by the cathepsin B inhibitor. These results indicate that this active peptide was produced by the degradation of cathepsin B. Reference Example 4 Inhibitory Effect of Cathepsin B on Proliferative Response of Human T Cells by Antigen In the presence of X-irradiated peripheral blood mononuclear cells (Rx-PBMC; cells that cannot proliferate but can present antigen), Human T cell clones (eg clones 2C5 and B
8) and the like show a high proliferative response by the vaccine, and are promoted by the peptide derived from the viral glycoprotein and the peptide derived from the nucleoprotein, respectively. In this system, the proliferative action of T cell clones by various proteases was examined. As a result, the response was suppressed in a dose-dependent manner by cysteine protease inhibitors, especially cathepsin B inhibitors, but Rx pretreated with the vaccine
-When PBMC is used as an antigen presenting cell (APC),
This proliferative response is not suppressed by cathepsin B inhibitors. Moreover, the proliferative response by the antigen peptide was not suppressed by the B inhibitor. In addition, the proliferative response of human T cell clones to the vaccine was suppressed by incubation with cathepsin B-specific antibodies, and cathepsin B specific substrates [eg Z-RR-MCA (benzyloxycarbonyl) -arginyl-arginyl-, Methylcoumarinamide), etc.] strongly suppressed the antigen response. MCA has no such effect. The above results suggest that cathepsin B is a protease involved in antigen processing. Reference Example 5 Effect of cathepsin B inhibitor on immune response in vivo In the presence or absence of animal cathepsin B, sensitization was performed with an active antigen peptide obtained by treating with cathepsin B together with an adjuvant. As a result, it was revealed that the immune response caused by the antigen peptide was not suppressed by the cathepsin B inhibitor. The results show that cathepsin B inhibitors do not interfere with processes of the immune response other than antigen processing. Reference Example 6 Binding of Antigen Peptide Processed by Cathepsin B to Class II MHC Molecule The amino acid sequences of cathepsin B and class II MHC were compared. As a result, one of the active sites of cathepsin B, VN
_217-223 (VANSWNT) is a Class II MH
Part of the binding domain of the Cβ chain, VN _57-63 (VAE
It was found to show homology with SWNS). Within this homologous sequence, especially the alanine and asparagine residues are identical to those of class II MHC molecules in cathepsin B, but these amino acid residues are replaced by lysine and glycine residues in other cathepsins, respectively. ing. This fact indicates that the antigenic peptides degraded by cathepsin B bind more specifically to class II MHC molecules. That is, the C-terminal domain sequences in antigenic peptides that show a common affinity with class II MHC molecules are defined by the substrate specificity of cathepsin B. Also, the majority of known antigenic peptide sequences isolated from class II MHC molecules [A. Y. Rudensky et al., Nature, 353, 622 (1991); ibid, 359, 429 (199).
2); K. Falkra et al., Ibid., Vol. 351, Vol. 2.
Page 90 (1992)] also supports the above consideration because it contains an affinity sequence for cathepsin B. Example 1 Effect of Cathepsin B Inhibitor on Antibody Production against HBs Antigen In Vivo A single intraperitoneal administration of 4 μg per HBs antigen (Kaketsuken Pharmaceutical Co., Ltd.) to BALB / c mice (8-10 weeks old). 2 hours before and after the inoculation with the control group, the cathepsin B inhibitor E-64 (Taisho Pharmaceutical Co., Ltd.) or CA-074 (200 μm
g / mouse) was intraperitoneally administered, and the anti-HBs antibody titer of the immunized administration group was measured by enzyme-linked immunosorbent assay (ELISA; manufactured by Abbott). The obtained results are shown in FIG. The data in the figure are expressed as mean value ± standard deviation (SD). In addition, ● indicates E-64a administration group, and Δ indicates CA-07.
4 administration groups, ◯ indicates a control group (physiological saline), and □ indicates a CA-074 continuous administration group for 14 days. As shown in the figure, in the control group, the anti-HBs antibody titer reached 200 mIU / ml 10 days after the inoculation, and reached the highest level (8
00 mIU / ml). On the other hand, in the administration group, anti-HB
S antibody production was suppressed and the antibody response was delayed. The degree of this suppression was maximum 14 days after inoculation. That is, the control group without administration of the inhibitor had an antibody titer of 500 mIU / ml, whereas CA-047 or E-64a.
Was 250 mIU / ml. Furthermore, when the cathepsin B inhibitor CA-074 was administered every day for 14 days after the antigen inoculation, the antibody production was significantly suppressed.
The antibody titer of 120 mIU / ml was only reached on the 4th day (the antibody titer of the non-inhibitor group n was 500 mIU / m).
l). Example 2 In vitro by restimulation of HBs antigen
Suppression of Proliferation Response of Sensitized Splenocytes by Cathepsin B Inhibitors As shown in Example 1, BALB / c mice were immunized with 4 μg of HBs antigen, and then sensitized splenocytes (1 ×
10 ⁵ ) was prepared and 1 g μ / ml of cathepsin E was prepared.
-64a or CA-047 and 4 μg / ml HB
S-antigen was added and incubated for 72 hours (HBs
Restimulation with antigen). After the reaction, the cells were mixed with 1 μCi of [ ³ H]-
After labeling with thymidine for 12 hours, [ ³ H] -thymidine incorporation was measured. The obtained results are shown in [Table 1].
The data in the table are represented by the average value ± SD (cpm) of 5 samples and the proliferation index (numerical values in parentheses). P <0.05; significant difference from control group (Student's t-test). As shown in Table 1, the sensitized spleen cells in the control group showed a proliferative response by restimulation with the HBs antigen, and [ ³ H] -thymidine incorporation was 4092 cpm. On the other hand, in the presence of the cathepsin B inhibitors CA-074 and E-64a, this cell proliferative response was 2112 cpm and 19 respectively.
It was remarkably suppressed to 80 cpm and decreased by 52% to 48% as compared with the proliferative response of the control group. Simultaneously with sensitization of mice with HBs antigen, E-64a or CA-047
The spleen cells obtained by administration of E. coli showed a very weak proliferative response to antigen restimulation of 550 cpm and 464 cpm, respectively. This proliferative response was further suppressed by adding E-64a or CA-074 during restimulation. Example 3 in v by restimulation of HBs antigen peptide
Inhibition of in vitro sensitized splenocyte proliferation response by cathepsin B inhibitor It is suggested that HBs antigen [Fig. 3] (SEQ ID NO: 3) is most likely to be degraded by cathepsin B between amino acid residues 79 and 80 due to the substrate specificity of cathepsin B. Although presumed, this binding was actually observed in vitro by a purified preparation of cathepsin B.
Decomposed in tro. Next, the peptide SR _64-79 (S consisting of 16 amino acid residues up to the arginine residues of amino acid residue numbers 64 to 79 of the N-terminal side of the HBs antigen molecule)
CPPICPGYRWMCLRR (sequence of amino acid residue numbers 64 to 79 of SEQ ID NO: 3), peptide SL _64-94 (S- _64-94 consisting of 31 amino acid residues of 64 to 94)
CPPICPGYRWMCLRRFIIFIFILL
CLIFL) (amino acid residue number 64 to SEQ ID NO: 3)
94 sequence) and a peptide FG _93-112 (FLLVLLDY) consisting of 20 amino acid residues _{93 to} 112.
QGMLPVCPLLPG) (sequences of amino acid residue numbers 93 to 113 of the sequence of SEQ ID NO: 3) were each automatically synthesized by an applied biosystem model A4311 peptide synthesizer according to a conventional method. For each 1 μg / ml of the obtained peptides, the proliferative response to HBs antigen-sensitized splenocytes was measured according to the method described in Example 2 [Table 1]. As shown in Table 1, peptide SR _64-79 showed the strongest proliferative response (256
8 cpm), but the peptides SL _64-94 and FG _93-112 of the other domains in the HBs antigen molecule.
Are extremely weak proliferative responses in the same system (16 each
26 cpm and 556 cpm). In addition, the proliferative response to peptide SR _64-79 is cathepsin B.
It was not inhibited at all by the inhibitor CA-074. From these results, it was concluded that the SR _64-79 sequence is one of the strongly antigenic peptides in the HBs antigen, and this fragment is generated by the degradation of the HBs antigen by cathepsin B. ◎

[Table 1] Example 4 Inhibitory Effect of Cathepsin B Inhibitors E-64d and CA-074 on Proliferative Response of Human T Cell Clones by Restimulation with Rabies Vaccine or Antigen Peptide (1) Peripheral Blood Treated with X-ray Irradiation (3000 rad) Human T cell clone 2C in the presence of mononuclear cells (Rx-PBMC; 1 × 10 ⁵ / well; cells that do not proliferate but have antigen-presenting ability and can stimulate T cells)
5 (3 × 10 ⁴ / well) complete medium (RPMI supplemented with 10% human AB serum) at various concentrations of 0.05-0.4 μg / ml rabies virus (Merieux, Lyon France) and 1.25, 2.5 And 5 μg of each concentration of cathepsin B inhibitor (E-64d or CA-074) were added, and the mixture was incubated at 37 ° C. in 5% carbon dioxide gas for 72 hours for reaction. Similar measurements were performed using Rx-PBMC (antigen-presenting state) pretreated with rabies vaccine. Before the end of reaction 1
1 μCi [ ³ H] -thymidine / well was incorporated for 6 hours, and the proliferative response was measured using [ ³ H] -thymidine of T cells as an index. The results obtained using E-64d and CA-074 as cathepsin B inhibitors are shown in FIG. 3 respectively.
And [Fig. 4]. As shown in [FIG. 4] and [FIG. 5], in the control sample to which the cathepsin B inhibitor was not added, 0.
Rabies vaccine at 2 μg / ml (Merieux, Ly
, France), and the proliferative response of this 2C5 clone was E-64d or CA-.
It was suppressed in a dose-dependent manner by the addition of 047. When pretreated Rx-PBMC was used, it was not suppressed by the cathepsin B inhibitor as shown by the dotted line. (2) Instead of the rabies vaccine of (1) above, a peptide ER _281-299 (EECLDALE) derived from a glycoprotein of rabies virus, which is an antigen peptide of the vaccine, is used.
STMTTKSVSFR) (SEQ ID NO: 4),
It measured according to the method of (1). As a result, Rx-PB
The growth of human T cell clone 2C5 in the presence of MC was promoted by both E-64 addition [Fig. 6] and CA-074 addition [Fig. 7]. The proliferative response by these antigenic peptides is cathepsin B inhibitor E-6.
It was not suppressed by 4d and CA-074. Symbols in FIGS. 4 to 6 are as follows. Δ Pretreated Rx-PBMC + 5 μg / ml inhibitor ○ Rx-PBMC + antigen + 1.25 μg / ml inhibitor x Rx-PBMC + antigen + 2.5 μg / ml inhibitor Rx-PBMC + antigen + 5 μg / ml inhibitor □ Rx-PBMC + Antigen Example 5 Inhibition of T Cell Proliferation by Cathepsin B Specific Antibody and Cathepsin B Substrate Human T cell clone 2C5 (3 × 10 ⁴ ) contains anti-cathepsin B antibody F (ab) ′ (Kominam at various concentrations.
i, E and Katanuna, N Journal of Biochemistry, Vol. 91, p. 67-71).
0 rad) treated peripheral blood mononuclear cells (Rx-PBM)
C; 1 × 10 ⁵ ) and 0.2 μg / ml of rabies vaccine were incubated for 72 hours. After reaction 1 μCi / w on cells
[ ³ H] -thymidine of ell was incorporated for 12 hours and collected, and the proliferation response was measured using [ ³ H] -thymidine incorporation as an index. As a result, the proliferative response of clone 2C5 to the rabies vaccine was confirmed by anti-cathepsin B antibody F (a
It was suppressed by co-incubation with b) '.
When a synthetic substrate for cathepsin B, Z-RR-MCA (benzyloxycarbonylarginylarginylmethylcoumarylamide) was added, the proliferative response was strongly inhibited [Fig. 8]. Example 6 Effect of cathepsin B inhibitor during sensitization on proliferation of sensitized splenocytes BALB in the presence or absence of cathepsin B inhibitor
/ C mouse splenocytes were sensitized with a rabies vaccine, and the resulting sensitized cells were measured for their proliferative response upon stimulation with various concentrations of the rabies vaccine and its antigenic peptide ER _281-299 . BALB / sensitized with rabies vaccine
C mouse splenocytes showed a strong proliferative response to in vitro vaccine or stimulation with the synthetic peptide ER _281-299 . On the other hand, CA-074 or E-
Cells sensitized with the rabies vaccine in the presence of 64d strongly suppressed the proliferative response of splenocytes to the vaccine or the antigenic peptide ER _281-299 in vitro [Fig. 9] and [Fig. 10]. Example 7 Cathepsin B Active Site and Class II MH
Homology of antigenic peptide binding domain of C molecule When sensitizing T cell clone 2C5 with rabies vaccine in the presence of Rx-PBMC, antigenic peptide S of HBs antigen
C-terminal peptide of R _64-79 (MCLRR or Y
RWMCLRR; 100 μg / ml)
Cell proliferation ([ ³ H] -thymidine incorporation) was 75% or 60% that of controls without these peptides.
Was suppressed to. This result indicates that these peptides inhibit antigen presentation by cathepsin B and also class II.
It has been shown to inhibit the binding of antigenic peptides to MHC molecules.

[Sequence list]

[Brief description of drawings]

FIG. 1 is a schematic diagram of the action of cathepsin B in antigen presentation.

FIG. 2 shows the amount of antibody produced against immunization with HBs antigen in the presence or absence of a cathepsin B inhibitor.

FIG. 3 shows the amino acid sequence of HBs antigen. Further, the portion corresponding to the antigen peptide SR _64-79 used in Example 3 was boxed.

FIG. 4 shows the proliferative response of T cell clones to antigen restimulation in the presence or absence of a cathepsin B inhibitor.

FIG. 5 shows the proliferative response of T cell clones to antigen restimulation in the presence or absence of a cathepsin B inhibitor.

FIG. 6 shows the proliferative response of T cell clones to antigen restimulation in the presence or absence of a cathepsin B inhibitor.

FIG. 7 shows the proliferative response of T cell clones to antigen restimulation in the presence or absence of a cathepsin B inhibitor.

FIG. 8 shows the proliferative response of T cell clones to antigen restimulation in the presence of anti-cathepsin B antibody or cathepsin B synthetic substrate.

FIG. 9 shows proliferative responses to antigen restimulation of spleen cells sensitized with or without a cathepsin B inhibitor.

FIG. 10 shows proliferative response to antigen restimulation of spleen cells sensitized with or without a cathepsin B inhibitor.

Claims (7)

[Claims] 1. An immunosuppressant containing an epoxysuccinic acid derivative. 2. The immunosuppressive agent according to claim 1, which contains an epoxysuccinic acid derivative having a cathepsin B inhibitory action. 3. The immunosuppressive agent according to claim 1, wherein the epoxysuccinic acid derivative is an epoxysuccinic acid ester derivative. 4. The immunosuppressive agent according to claim 1, wherein the epoxysuccinic acid derivative is an epoxysuccinamide derivative. 5. The epoxysuccinamide derivative is N- (L
The immunosuppressant according to claim 4, which is -3-trans-n-propylcarbamoyloxirane-2-carbonyl) -L-isoleucyl-L-proline. 6. The epoxysuccinamide derivative is N-
(L-3-trans-ethoxycarbonyloxirane-
The immunosuppressant according to claim 4, which is 2-carbonyl) -L-leucine-3-methylbutyramide. 7. The immunosuppressive agent according to claim 1, which is a therapeutic agent for autoimmune diseases.