JP2002114800A - Apoptosis controlling peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a peptide which provides a peptide having new inhibitory activity against TNF, TRAIL and FasL and its pharmacologically acceptable salt, inhibits apoptosis, an inflammation, etc., induced by TNF, TRAIL and FasL and is useful for treating diseases caused by TNF, TRAIL and FasL, and its pharmacologically acceptable salt. SOLUTION: This peptide contains a human-derived specific sequence and has TNP inhibitory activity and its pharmacologically acceptable salt. This peptide contains a human-derived specific sequence different from the sequence and has TRAIL inhibitory activity and its pharmacologically acceptable salt. This peptide contains a human-derived specific sequence different from the two sequences and has FasL inhibitory activity and its pharmacologically acceptable salt.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel TNF, TRAIL,
The present invention relates to a peptide having FasL inhibitory activity and a physiologically acceptable salt thereof. More specifically, the present invention relates to TN
F, TRAIL, inhibits apoptosis and inflammation induced by FasL, which causes systemic lupus erythematosus, Hashimoto's disease, rheumatoid arthritis, graft-versus-host disease, Sjogren's syndrome, pernicious anemia, Addison's disease, scleroderma, Autoimmune diseases such as Goodpasture's syndrome, Crohn's disease, autoimmune hemolytic anemia, natural infertility, myasthenia gravis, multiple sclerosis, Basedow's disease, idiopathic thrombocytopenic purpura, and insulin-dependent diabetes; allergy; Atopy; arteriosclerosis;
Myocarditis; cardiomyopathy; glomerulonephritis; aplastic anemia; fulminant hepatitis, chronic hepatitis, viral hepatitis, alcoholic hepatitis, etc .; AIDS; rejection after organ transplantation; septic shock;
The present invention relates to a peptide useful for treating congestive heart failure; myelodysplastic syndrome; cancer and the like, and a pharmaceutically acceptable salt thereof.

[0002]

2. Description of the Related Art Apoptosis is a spontaneous death necessary to maintain homeostasis of a living body, and its breakdown leads to serious diseases such as cancer and autoimmune diseases (Annu Rev Immunol, 17, 221-25
3, 1999). Apoptosis is also triggered by external factors such as radiation and drugs, but TNF, T
RAIL, FasL, etc. have been reported (Pharm Acta Helv, 74, 2
81-286, 2000; Exp Cell Res, 256, 58-66, 2000). They bind to specific receptors on target cells and induce apoptosis (TIBS, 24 February, 47-5
3, 1999).

[0003] On the other hand, overproduction of TNF, TRAIL, and FasL is caused by rheumatoid arthritis, multiple sclerosis.
(Multiple sclerosis), AIDS (Acquired immune deficie)
ncysyndrome), cancer, septic shock, congestive heart failure, aplastic anemia, myelodyspl
(Microsc Res Tech, 50, 229-235, 200)
0).

[0004] Humanized immunoglobulins that specifically react with FasL are known, and FasL represented by apoptosis is known.
Has been shown to inhibit the physiological response between
O98 / 10070).

[0005] A novel pharmaceutical composition containing an anti-Fas antibody as an active ingredient, which is useful as a therapeutic agent for diseases caused by abnormalities of the Fas / FasL system, is known (JP-A-2000-1693).
93).

[0006] Death effector domain combined with Caspase-8
Is known to have apoptosis control ability (JP-A-2000-226400).

MORT-1, which binds to the intracellular domain of Fas,
Alternatively, a protein that binds to TRADD and modulates the effect of FasL or TNF, and a peptide that inhibits the activity of the protein is known (Japanese Patent Application Laid-Open No. H11-509422).

[0008] An inhibitor or preventive agent for apoptosis caused by the interaction between FasL on cytotoxic T cells and Fas on an organ cell membrane containing epidermal growth factor (EGF) as an active ingredient is known. (JP-A-10-19498
8).

[0009] Synthetic peptides obtained by searching for a TNF-binding functional part of a natural TNF-binding protein are known, and mouse L-
It has been shown to inhibit the cytotoxicity of TNF on M cells (JP-A-5-194594).

7 consisting of triazepine or diazepine
An apoptosis inhibitor characterized by being a condensed ring compound of a membered ring and a 5-membered ring containing 2 to 4 nitrogen atoms has been known (JP-A-11-228576).

Apoptosis inhibitors represented by antibodies are
Since the protein has a high specificity but a large molecular weight, it has problems in antigenicity, administration method and stability. Synthetic peptides obtained by searching for the TNF-binding functional part of the natural TNF-binding protein have reduced efficacy due to competition with the natural TNF-binding protein,
There are problems such as a short residence time in blood. In addition, since apoptosis inhibitors of low molecular weight organic compounds inhibit the basic pathway of apoptosis, side effects due to low selectivity are problematic.

[0012]

Accordingly, the present invention provides a novel peptide having TNF, TRAIL, and FasL inhibitory activity and a pharmaceutically acceptable salt thereof, and further provides TNF, TRA
Inhibits apoptosis and inflammation induced by IL and FasL, which causes systemic lupus erythematosus, Hashimoto's disease, rheumatoid arthritis, graft-versus-host disease, Sjogren's syndrome, pernicious anemia, Addison's disease, scleroderma, Goodpasture Autoimmune diseases such as syndrome, Crohn's disease, autoimmune hemolytic anemia, natural infertility, myasthenia gravis, multiple sclerosis, Basedow's disease, idiopathic thrombocytopenic purpura, insulin-dependent diabetes mellitus; allergy; atopy; Arteriosclerosis; myocarditis; cardiomyopathy; glomerulonephritis; aplastic anemia; fulminant hepatitis;
Peptides useful for treating hepatitis such as chronic hepatitis, viral hepatitis, alcoholic hepatitis, etc .; AIDS; rejection after organ transplantation; septic shock; congestive heart failure; myelodysplastic syndrome; The purpose is to give a salt.

[0013]

According to the present invention, the above object is achieved by the following amino acid sequence (1): His Cys Leu SerCy
s Ser Lys Cys Arg Lys Glu Thr Gly Gln (SEQ ID NO:
Peptide having TNF inhibitory activity comprising the sequence represented by 1) and a pharmaceutically acceptable salt thereof, and the following amino acid sequence (2): Phe Cys Leu Arg Cys Thr Arg Cys
TR represented by Asp Ser Gly GluVal Glu (SEQ ID NO: 2)
Peptides having AIL inhibitory activity and physiologically acceptable salts thereof, and the following amino acid sequence (3): Lys
Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly L
This is achieved by a peptide having FasL inhibitory activity represented by eu (SEQ ID NO: 3) and a pharmaceutically acceptable salt thereof.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, various amino acid residues are described by the following abbreviations. Ala: L-alanine residue Arg: L-arginine residue Asn: L-asparagine residue Asp: L-aspartic acid residue Cys: L-cysteine residue Gln: L-glutamine residue Glu: L-glutamic acid residue Gly: glycine residue His: L-histidine residue Ile: L-isoleucine residue Leu: L-leucine residue Lys: L-lysine residue Met: L-methionine residue Phe: L-phenylalanine residue Pro: L -Proline residue Ser: L-serine residue Thr: L-threonine residue Trp: L-tryptophan residue Tyr: L-tyrosine residue Val: L-valine residue

Further, in the present specification, the amino acid sequence of the peptide is described in a conventional manner such that the N-terminal amino acid residue is located on the left side and the C-terminal amino acid residue is located on the right side.

[0016] The peptide of the present invention is synthesized by a usual peptide synthesis method. For example, it is prepared by a solid phase synthesis method or a liquid phase synthesis method, but the solid phase synthesis method is simple in operation [for example, “Seizoku Chemistry Experimental Course 2 Protein Chemistry (2)” edited by The Biochemical Society of Japan (Showa 62) May 20, 2000, issued by Tokyo Chemical Co., Ltd.), pages 641-694.

In the preparation of the peptide of the present invention by the solid phase synthesis method, for example, the amino acid corresponding to the C-terminus of the peptide of interest is converted into a polymer insoluble in a reaction solvent such as a styrene-divinylbenzene copolymer. Α-COO having
An amino group other than the α-COOH group possessed by the amino acid or peptide fragment, wherein the amino acid is linked to the amino acid in the direction of the N-terminus of the target peptide; Sequentially protecting and protecting the functional group and condensing and bonding, and removing the protecting group of an amino group forming a peptide bond such as an α-amino group in the bonded amino acid or peptide fragment in order. To form a peptide chain corresponding to the target peptide, and then remove the peptide chain from the polymer, and remove the protecting group from the protected functional group. This is done by obtaining the peptide, which is then purified. Here, the elimination of the peptide chain from the polymer and the removal of the protecting group are preferably performed simultaneously using trifluoroacetic acid from the viewpoint of suppressing side reactions. Further, it is effective that the obtained peptide is purified by reversed-phase liquid chromatography or gel permeation chromatography.

The salt of the peptide of the present invention is prepared by utilizing a usual salt formation reaction.

The amino acid sequence of the peptide provided by the present invention is (1): His Cys Leu Ser Cys Ser Lys Cys A
rg Lys Glu Thr Gly Gln (SEQ ID NO: 1), (2): Phe C
ys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu
(SEQ ID NO: 2), (3): Lys Cys Arg Arg Cys Arg Leu
Given as Cys Asp Glu Gly His Gly Leu (SEQ ID NO: 3).

[0020] The peptide of the present invention comprises the aforementioned SEQ ID NOS: 1 to 3
Wherein the amino terminal may be substituted with an acetyl group, a methoxycarbonyl group, a butoxycarbonyl group, a peptide of 1 to 5 residues, or the like, and the carboxy terminal may be an amide group, It may be substituted with a methyl ester group, a butyl ester group, a peptide having 1 to 5 residues, or the like. Further, each amino acid residue may be subjected to homology substitution based on the properties of the side chain functional group. For example, Leu is Ile, Val, Ser is Thr, Glu is Asp
In addition, Gln can be replaced with Asn, Lys can be replaced with Arg, respectively.
The converse is also interchangeable. Further, SEQ ID NOs: 1 to 3
In the amino acid sequence described in any of the above, 1 to 3 amino acid residues, preferably 1 or 2 amino acid residues, more preferably 1 amino acid residue may be added, , 1 to 3 amino acid residues, preferably 1 or 2 amino acid residues, more preferably 1 amino acid residue may be deleted. Even then, induction of apoptosis or inflammation is inhibited.

The peptide of the present invention may form a pharmaceutically acceptable salt such as hydrochloric acid, sulfuric acid, phosphoric acid, lactic acid, tartaric acid, maleic acid, fumaric acid, oxalic acid, and the like. Salts with acids such as malic acid, citric acid, oleic acid, palmitic acid; salts with alkali or alkaline earth metals such as sodium, potassium, calcium or with hydroxides or carbonates of aluminum; triethylamine, benzyl Amines, diethanolamine, t-butylamine, dicyclohexylamine,
And salts with arginine and the like.

The TNF, TRAIL, and FasL inhibitory effects of the peptide of the present invention can be measured by a usual method for measuring these activities. For example, TNF activity is measured by measuring (1) cytotoxic activity targeting mouse L cells, (2) lipoprotein lipase inhibitory activity, and (3) proliferation promoting activity on fibroblasts [eg, Nakajima Hikami et al. New Basic Biochemistry Experiment Method 6 "A Method for Measuring Biological Activity" (April 3, 1988)
0 Maruzen Co., Ltd.), pages 267-272]. In addition, TNF activity was measured in mouse L929 cells and NIH
It can be measured by inducing apoptosis of 3T3 cells. TR
AIL activity can be measured by inducing apoptosis on mouse L929 cells. The activity of FasL can be measured by inducing apoptosis in mouse L929 cells or NIH3T3 cells into which the Fas gene has been introduced (for example, EMBO J, 13, 4587-4).
596, 1994.). For the detection of apoptosis induction, a method for detecting a change in the membrane structure such as a nucleic acid staining reagent such as propidium iodide (PI) or Hoechst 33258 or a fluorescently labeled annexin V is preferably used. Among them, propidium iodide (PI) and FITC-labeled annexin V are particularly preferred.

Therapeutic methods using the peptide of the present invention include, for example, systemic administration such as intravenous administration, subcutaneous administration, intraperitoneal administration, transdermal administration, and oral administration, and catheterization at a diseased site such as intraarticular administration. Examples include a method of injecting the peptide of the present invention using an injection needle.

The dose of the peptide of the present invention for effective expression of activity is usually 0.01 μg / kg to 2 g / kg (adult), preferably 0.01 μg / kg to 200 m.
g / kg (adult).

As the administration form, a solution obtained by dissolving the peptide in a pharmaceutically acceptable solution such as 5% glucose solution or physiological saline is preferable, and the solution may be any of various pharmacologically acceptable additives. It may contain an agent. Further, the peptides can be encapsulated or liposomal.

Production of Antibodies Various host animals can be immunized by injecting a composition comprising the peptide of the present invention comprising the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 and / or SEQ ID NO: 3. I can do it. Host animals include rabbits, mice, guinea pigs, rats, sheep, goats and the like. Various adjuvants can be used to enhance the immune response depending on the host species, for example, Freund (complete or incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin , Pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol and potentially useful human adjuvants such as BCG (sterile tuberculosis bovine tuberculosis) and Corynebacterium parvum And the like, but are not limited thereto.

Antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, F (ab ') ₂
Fragments, molecules produced using Fab expression libraries, and the like.

A monoclonal antibody, which is a homogeneous collection of antibodies against a specific antigen, uses the above-described peptide,
Standard hybridoma technology (Kohler et al .: Nature, 256:
495, 1975; Kohler et al .: Eur. J. Immunol., 6: 511, 197.
6; Kohler et al .: Eur. J. Immunol., 6: 292, 1976; Hummerling et al .: In: Monoclonal Antibodies and T CellHy.
bridomas, Elsevier, NY, 1981; U.S. Patent 4,376,11
0; Cosboa et al .: Immunology Today, 4:72, 1983; Kohle et al .: Proc. Natl. Acad. Sci. USA, 80: 2026,1983; Kohle et al .: Monoclonal Antibodies and Cancer Therapy, Alan
R. Liss, Inc., pp. 77-96, 1983). Such an antibody may be an Ig
It can be selected from any immunoglobulin class, including G, IgM, IgE, IgA, IgD and subclasses thereof. Hybridomas producing monoclonal antibodies may be cultured either in vitro or in vivo.

Antibodies can be used, for example, to detect the presence of an antigen in a biological sample as part of a diagnostic assay, and for assessing the efficacy of a medical treatment by other therapeutic methods. Can also be used.

Immunoassays A further embodiment of the invention comprises SEQ ID NO: 1, SEQ ID NO:
The present invention relates to the field of immunoassay using an antibody that specifically recognizes, as an antigen, the peptide of the present invention containing the amino acid sequence represented by SEQ ID NO: 2 and / or SEQ ID NO: 3.

The peptide containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 and / or SEQ ID NO: 3, which is specifically recognized as the antigen, may have an amino terminal at an acetyl group, a methoxycarbonyl group or a butoxy group, if desired. It may be substituted with a carbonyl group, a peptide having 1 to 5 residues or the like, or may be substituted with an amide group, a methyl ester group, a butyl ester group, a peptide having 1 to 5 residues, or the like. Further, each amino acid residue may be subjected to homology substitution based on the properties of the side chain functional group.
For example, Leu is Ile, Val, Ser is Thr, Glu is Asp, G
ln can be substituted for Asn and Lys can be substituted for Arg, and vice versa. Furthermore, in the amino acid sequence of any one of SEQ ID NOs: 1 to 3, 1 to 3 amino acid residues, preferably 1 or 2 amino acid residues, more preferably 1 amino acid residue May be added, and 1 to 3 amino acid residues, preferably 1 or 2 amino acid residues, more preferably 1 amino acid residue may be deleted. Even then, induction of apoptosis or inflammation is inhibited.

Further, the peptide may form a pharmaceutically acceptable salt. Examples of the salt include hydrochloric acid, sulfuric acid, phosphoric acid, lactic acid, tartaric acid, maleic acid, fumaric acid, oxalic acid, and the like. Salts with acids such as malic acid, citric acid, oleic acid, palmitic acid; salts with alkali or alkaline earth metals such as sodium, potassium, calcium or with hydroxides or carbonates of aluminum; triethylamine, benzyl Examples include salts with amines, diethanolamine, t-butylamine, dicyclohexylamine, arginine and the like.

[0033] In one embodiment, the immunoassay comprises:
It is an enzyme-linked immunosorbent assay (ELISA). In this type,
The sample and predetermined reagents are contained within the wells of a microtiter well plate, and the assay is initiated by adding to the wells a detection reagent suitable for detecting the enzyme label. Such assays are known to those of skill in the art, and background information relevant to the immunoassay techniques can be found in The Immunoassay Handb
ook ', (Wild, DG Ed, Stockton Press, New York,
[1994]).

Suitable exemplary enzyme labels include, but are not limited to, alkaline phosphatase, β-galactosidase, horseradish peroxidase, malate dehydrogenase and glucose-6-phosphate dehydrogenase. Wasabi peroxidase
Particularly preferred enzyme labels for use in the enzyme immunoassay of the present invention.

In a further different embodiment, the immunoassay comprises:
Radioisotopes that are radioimmunoassays and that are suitable for use in the assays of the invention include β-emitting isotopes, such as tritium, and iodine, which emits Auger electrons.
125 are included.

In yet another embodiment, the immunoassay is a fluorescent immunoassay and the appropriate fluorescent label is selected from fluorescein, rhodamine and cyanine dyes.

EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples. In addition,
The present invention is not limited by these examples. (Example 1) Formula (1): His Cys Leu Ser Cys Ser Lys Cys Arg Lys G
peptide containing the amino acid sequence represented by lu Thr Gly Gln (SEQ ID NO: 1) His Cys Leu Ser Cys Ser LysCys Arg L
ys Glu Thr Gly Gln-NH ₂ was synthesized by a solid phase synthesis method using an automatic peptide synthesizer. That is, 4- (2 ′,
Styrene-divinylbenzene copolymer having 4'-dimethoxyphenyl-fluorenylmethoxycarbonyl) -aminomethyl) -phenoxyacetamido-ethyl group at a ratio of 0.62 mmol / g (resin) [Constitution of styrene and divinylbenzene Using 0.1 mmol of a granular resin (molar ratio: 99: 1, Fmoc amide resin manufactured by Applied Biosystems, USA), the corresponding amino acids are successively bonded from the carboxyl terminal to the amino terminal of the target peptide. Was. In the coupling reaction, as an amino acid, N ^α -9- (fluorenylmethoxycarbonyl) -N ^γ -manufactured by Applied Biosystems, USA
Trityl-L-glutamine [Fmoc glutamine], N
^α -9- (fluorenylmethoxycarbonyl) -glycine [Fmoc glycine], N ^α -9- (fluorenylmethoxycarbonyl) -Ot-butyl-L-threonine [Fmoc threonine], N ^α -9- (fluorenyl methoxycarbonyl)-.gamma.-butyl -L- glutamic acid [Fmoc glutamic acid], N ^{alpha-9-(fluorenyl} methoxy carbonyl) -N ^{epsilon-t-butoxycarbonyl} -L- lysine [Fmoc lysine], N ^{alpha-9-(fluorenyl} methoxy carbonyl) -N ^G - (2,2,5,7,
8-pentamethylchroman-6-sulfonyl) -L-
Arginine ^{[Fmoc-arginine],} N α -9- (fluorenyl methoxy carbonyl) -S- trityl -L- cysteine [Fmoc ^cysteine], N α -9- (fluorenyl methoxy carbonyl) -O-t-butyl - L-serine [Fmoc serine], N ^α -9- (fluorenylmethoxycarbonyl) -L-leucine [Fmoc leucine], N ^α -9- (fluorenylmethoxycarbonyl)-
N ^Im -trityl-L-histidine [Fmoc histidine] was used at 1 mmol for each conjugation step.

The resulting peptide resin was mixed with 7.5% phenol, 2.5% ethanedithiol, 5% water and 5%
Treated with 10 ml of trifluoroacetic acid containing 3% thioanisole for 3 hours. The resulting solution was added to diethyl ether, and the resulting precipitate was washed several times with diethyl ether to deprotect the peptide and to remove it from the resin. The crude product was purified with a PD10 column (Amersham Pharmacia Japan) to obtain a peptide. AKTAexpl manufactured by Pharmacia Biotech Co., Ltd.
orer10XT [column: Novapack C18 manufactured by Millipore Waters Co., Ltd. 3.9 mmφ × 150 mm,
Mobile phase: mixed solvent of acetonitrile and water containing 0.05% by volume of trifluoroacetic acid (acetonitrile concentration of 3
(Linearly changed from 5% by volume to 50% by volume in 0 minutes) and a flow rate of 1.0 ml / min].
A single peak was shown at 6 min. The molecular weight of the purified peptide determined by the FAB method mass spectrum was 1579 (theoretical value: 1578.86).

(Example 2) Formula (2): Phe Cys Leu Arg Cys Thr Arg Cys Asp Ser G
ly Glu Peptide containing an amino acid sequence represented by Val Glu (SEQ ID NO: 2) Phe Cys Leu Arg Cys Thr ArgCys Asp S
er Gly Glu Val Glu-NH ₂ and formula (3): Lys Cys Arg
A peptide represented by Arg Cys ArgLeu Cys Asp Glu Gly His Gly Leu (SEQ ID NO: 3) was synthesized in the same manner as in Example 1. However, in the case of the peptide containing the amino acid sequence represented by the formula (2), in addition to the amino acids shown in Example 1 in the binding reaction, N ^α -9- (fluorenyl) manufactured by Applied Biosystems, USA Methoxycarbonyl) -γ-butyl-L-glutamic acid [Fmoc glutamic acid], N ^α- (fluorenylmethoxycarbonyl)-
L-valine [Fmoc valine], N ^α -9- (fluorenylmethoxycarbonyl) -β-butyl-L-aspartic acid [Fmoc aspartic acid], N ^α- (fluorenylmethoxycarbonyl) -L-phenylalanine [ F
moc phenylalanine] was used at 1 mmol for each coupling step. Equation (3) in the case of a peptide represented ^by, 4- (N α -9- (fluorenyl methoxy carbonyl) -L- leucine) - oxymethyl - phenoxy - 0.74 mmol / g (resin methyl A) a granular resin comprising a styrene-divinylbenzene copolymer [constituent molar ratio of styrene and divinylbenzene: 99: 1] [manufactured by Applied Biosystems, USA;
HMP leucine] 0.1 mmol was used.

Phe Cys Leu Arg Cys Thr Arg Cys Asp Se
The purified peptide represented by _{r Gly Glu Val Glu-NH 2} ,
A single peak was shown at 14.3 min, and the molecular weight of the purified peptide was 1617 (theory: 1616.86).
In the purified peptide represented by SEQ ID NO: 3, 12.9 mi
n indicates a single peak, and the molecular weight of the purified peptide is 1
646 (theory: 1645.96).

(Test Example 1) 24-hole plate (NUNC)
50,000 L929 cells dispersed in Eagle's medium (10% FCS / E-MEM) containing 10% fetal bovine serum, or Dulbecco's modified Eagle medium containing 10% fetal bovine serum (10% FCS / D-ME
M) of 50,000 NIH3T3 cells dispersed in 1 ml each
Was dispensed. After culturing at 37 ° C. for 24 hours in 5% CO ₂ to allow the cells to adhere, a peptide buffer of the present invention in a phosphate buffer (PBS: 10 mM, containing 0.15 M sodium chloride, pH 7.4) was added. 100μl or 200μ
l, and rhTNFα (R & D Systems) or rhTRAIL (R & D Systems) or rhFas
10 μl of L (R & D Systems) in PBS was added to each well. When using rhFasL (R & D Systems) or rhTRAIL, an anti-His tag antibody (R & D Systems) was further added to a final concentration of 10 μg / ml. When NIH3T3 cells were used, cycloheximide was further added to a final concentration of 5 μg / ml. 37 ° C, 5% CO ₂
After culturing for 12 hours, propidium iodide (PI)
Was added to the medium to a final concentration of 1 μg / ml,
Furthermore, it was left still for 15 minutes at 37 ° C. and 5% CO ₂ . The fluorescence of PI was observed with a confocal laser microscope, and cells induced to apoptosis were identified. The ratio of cells induced to apoptosis to the total number of cells in five randomly selected visual fields was calculated and defined as the apoptosis induction rate.

RhTNFα was added to L929 cells by 50
The apoptosis induction rate when using ng / ml and adding PBS instead of the peptide solution was 70.3%, whereas His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu.
Thr Gly Gln-NH ₂ peptide at a final concentration of 100 μg / m
50.7% when added to give l, SEQ ID NO:
63.7% when Peptide 3 was added to a final concentration of 100 μg / ml, Phe Cys Leu Arg Cys Thr
When the peptide of Arg Cys Asp Ser Gly Glu Val Glu-NH ₂ is added to a final concentration of 100 μg / ml, 6
It was 5.6%.

Similarly, rhTRA was used for L929 cells.
The apoptosis induction rate was 55.4 when IL was used at 100 ng / ml and PBS was added instead of the peptide solution.
%, Whereas His Cys Leu Ser Cys Ser Lys Cys
Arg Lys Glu Thr Gly Gln-NH ₂ peptide at final concentration 2
52.0 when added to be 00 μg / ml
%, Phe Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Gl
u Peptide of Val Glu-NH ₂ at final concentration of 200 μg / ml
It was 46.6% when added so as to be.

RhFasL was used at 100 ng / ml for NIH3T3 cells, and PF was used instead of the peptide solution.
Apoptosis induction rate when BS was added was 34.1%, whereas His Cys Leu Ser Cys Ser Lys Cys Arg Ly
s Glu Thr Gly Gln-NH ₂ peptide at a final concentration of 200μ
g / ml, 29.9% when the peptide of SEQ ID NO: 3 is added to a final concentration of 200 μg / ml, Phe Cys Leu Arg Cy
It was 25.1% when the peptide of s Thr Arg Cys Asp Ser Gly Glu Glu Val Glu-NH ₂ was added to a final concentration of 200 μg / ml.

[0045]

As is clear from the above examples, the novel peptides provided by the present invention are TNF, TRAIL, Fa
It can effectively inhibit sL-induced apoptosis induction, which causes systemic lupus erythematosus, Hashimoto's disease, rheumatoid arthritis, graft-versus-host disease, Sjogren's syndrome, pernicious anemia, Addison's disease, scleroderma, Good Autoimmune diseases such as pasture syndrome, Crohn's disease, autoimmune hemolytic anemia, spontaneous infertility, myasthenia gravis, multiple sclerosis, Basedow's disease, idiopathic thrombocytopenic purpura, insulin-dependent diabetes; Atopy; arteriosclerosis;
Myocarditis; cardiomyopathy; glomerulonephritis; aplastic anemia; fulminant hepatitis, chronic hepatitis, viral hepatitis, alcoholic hepatitis, etc .; AIDS; rejection after organ transplantation; septic shock;
It is useful as a therapeutic agent for congestive heart failure; myelodysplastic syndrome;

[0046]

[Sequence List] SEQUENCE LISTING <110> Foundation for Nara Institute of Science and Technology <120> apoptosis-inhibited peptide <130> 174097 <140><141><160> 3 <170> PatentIn Ver. 2.1 <210> 1 <211> 14 <212> PRT <213> human <400> 1 His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Thr Gly Gln 1510 <210> 2 <211> 14 <212> PRT <400> human > 2 Phe Cys Leu Arg Cys Thr Arg Cys Asp
Ser Gly Glu Val Glu 15
10 <210> 3 <211> 14 <212> PRT <213> human <400> 3 Lys Cys Arg Arg Cys Arg Leu Cys Asp
Glu Gly His Gly Leu 15
10

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) A61P 5/14 A61P 5/40 5/40 7/04 7/04 7/06 7/06 9/00 9/00 9 / 10 101 9/10 101 13/12 13/12 15/08 15/08 17/00 17/00 19/02 19/02 21/04 21/04 25/00 25/00 29/00 29/00 101 101 31/04 31/04 31/12 31/12 31/18 31/18 35/00 35/00 37/00 37/00 37/02 37/02 37/08 37/08 43/00 105 43/00 105 C07K 16/44 C07K 16/44 G01N 33/53 D G01N 33/53 33/577 A 33/577 C12P 21/08 // C12P 21/08 A61K 37/02 F term (reference) 4B064 AG27 CA10 CA20 CC24 DA13 4C084 AA07 BA01 BA17 BA18 DA58 ZA022 ZA362 ZA532 ZA552 ZA662 ZA752 ZA812 ZA892 ZA942 ZA962 ZB072 ZB112 ZB152 ZB212 ZB262 ZB332 ZC062 ZC082 ZC352 ZC552 4H045 AA11 AA30 BA16 BA17 DA33

Claims (21)

[Claims] 1. The following amino acid sequence: His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Thr Gl
y Gln (SEQ ID NO: 1) Phe Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Va
l Glu (SEQ ID NO: 2) Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gl
y Leu (SEQ ID NO: 3), or a sequence in which 1 to 3 arbitrary amino acids have been added to the amino acid sequence, a sequence in which 1 to 3 amino acids have been deleted from the amino acid sequence, or the amino acid A peptide comprising a sequence having a homologous substitution in the sequence, the peptide inhibiting induction of apoptosis or inflammation, and a pharmaceutically acceptable salt thereof. 2. The following amino acid sequence: His Cys Leu Ser
Cys Ser Lys Cys Arg Lys Glu Thr Gly Gln (SEQ ID NO:
1) including a sequence in which 1 to 3 arbitrary amino acids have been added to the amino acid sequence, a sequence in which 1 to 3 amino acids have been deleted from the amino acid sequence, or a sequence in which homology has been substituted in the amino acid sequence. A peptide, which inhibits induction of apoptosis or inflammation by TNF, and a pharmaceutically acceptable salt thereof. 3. The following amino acid sequence: Phe Cys Leu Arg
Cys Thr Arg Cys Asp Ser Gly Glu Val Glu (SEQ ID NO:
2) including a sequence in which 1 to 3 arbitrary amino acids have been added to the amino acid sequence, a sequence in which 1 to 3 amino acids have been deleted from the amino acid sequence, or a sequence in which the amino acid sequence has been homologously substituted. A peptide, TRAIL
And a pharmaceutically acceptable salt thereof. 4. The following amino acid sequence: Lys Cys Arg Arg
Cys Arg Leu Cys Asp Glu Gly His Gly Leu (SEQ ID NO:
3), including a sequence in which 1 to 3 arbitrary amino acids are added to the amino acid sequence, a sequence in which 1 to 3 amino acids have been deleted from the amino acid sequence, or a sequence in which homology has been substituted in the amino acid sequence. A peptide which inhibits induction of apoptosis or inflammation by FasL, and a pharmaceutically acceptable salt thereof. 5. The amino terminal of the amino acid sequence is an acetyl group, a methoxycarbonyl group, a butoxycarbonyl group,
The peptide according to any one of claims 1 to 4, which is substituted with a peptide having 1 to 5 residues, and a pharmaceutically acceptable salt thereof. 6. The amino acid sequence wherein the carboxy terminus is an amide group, a methyl ester group, a butyl ester group,
The peptide according to any one of claims 1 to 4, which is substituted with a peptide having a residue, and a pharmaceutically acceptable salt thereof. 7. The salt of the pharmaceutically acceptable peptide according to claim 1, which is formed together with an acid. 8. The method according to claim 1, wherein the acid is hydrochloric acid, sulfuric acid, phosphoric acid, lactic acid, tartaric acid, maleic acid, fumaric acid, oxalic acid, malic acid,
8. A citric acid, oleic acid or palmitic acid.
A pharmaceutically acceptable salt of the peptide according to the above. 9. The pharmaceutically acceptable salt of a peptide according to claim 1, which is formed together with an alkali metal or alkaline earth metal hydroxide or carbonate. 10. The pharmaceutically acceptable salt of the peptide according to claim 10, wherein the alkali metal or alkaline earth metal is sodium, potassium, or calcium. 11. A pharmaceutically acceptable salt of a peptide according to claim 1, which is formed together with an aluminum hydroxide or carbonate. 12. Triethylamine, benzylamine,
The pharmaceutically acceptable peptide salt according to any one of claims 1 to 4, which is formed together with diethanolamine, t-butylamine, dicyclohexylamine, and arginine. 13. Systemic lupus erythematosus, Hashimoto's disease, rheumatoid arthritis, graft-versus-host disease, Sjogren's syndrome, pernicious anemia, Addison's disease, scleroderma, Goodpasture's syndrome, Crohn's disease, autoimmune hemolytic anemia, natural Autoimmune diseases such as infertility, myasthenia gravis, multiple sclerosis, Basedow's disease, idiopathic thrombocytopenic purpura, and insulin-dependent diabetes; allergy; atopy; arteriosclerosis; myocarditis; cardiomyopathy; glomerulonephritis Aplastic anemia; fulminant hepatitis,
13. Hepatitis such as chronic hepatitis, viral hepatitis, alcoholic hepatitis, etc .; AIDS; rejection after organ transplantation; septic shock; congestive heart failure; myelodysplastic syndrome; Or a pharmaceutically acceptable salt thereof. 14. An antibody that specifically recognizes the peptide according to any one of claims 1 to 13 and a pharmaceutically acceptable salt thereof as an antigen. 15. The method according to claim 1, which is a monoclonal antibody.
5. The antibody according to 4. 16. The antibody used in claim 14 and claim 1.
An immunoassay method, which is the antibody according to 5. 17. an enzyme immunoassay, a radioimmunoassay,
17. The immunoassay according to claim 16, wherein any one of a fluorescence immunoassay is selected. 18. Systemic lupus erythematosus, Hashimoto's disease, rheumatoid arthritis, graft-versus-host disease, Sjogren's syndrome, pernicious anemia, Addison's disease, scleroderma, Goodpasture's syndrome, Crohn's disease, autoimmune hemolytic anemia, natural Autoimmune diseases such as infertility, myasthenia gravis, multiple sclerosis, Basedow's disease, idiopathic thrombocytopenic purpura, and insulin-dependent diabetes; allergy; atopy; arteriosclerosis; myocarditis; cardiomyopathy; glomerulonephritis Aplastic anemia; fulminant hepatitis,
13. Hepatitis such as chronic hepatitis, viral hepatitis, alcoholic hepatitis, etc .; AIDS; rejection after organ transplantation; septic shock; congestive heart failure; myelodysplastic syndrome; An antibody that specifically recognizes any one of the peptides described above and a pharmaceutically acceptable salt thereof as an antigen. 19. The method according to claim 1, which is a monoclonal antibody.
9. The antibody according to 8. 20. The antibody according to claims 18 and 1,
An immunoassay, which is the antibody according to 9. 21. an enzyme immunoassay, a radioimmunoassay,
21. The immunoassay according to claim 20, wherein any one of a fluorescent immunoassay is selected.