JP4366455B2 - Apoptosis inhibitor peptide - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel peptide and a salt thereof having an activity of inhibiting apoptosis and inflammation. In particular, the present invention inhibits apoptosis and inflammation induced by TNF, TRAIL, FasL, etc., and causes autoimmune diseases (for example, 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, spontaneous infertility, myasthenia gravis, multiple sclerosis, Basedoh's disease, idiopathic thrombocytopenic purpura ), Allergy, atopy, arteriosclerosis, myocarditis, cardiomyopathy, glomerulonephritis, aplastic anemia, hepatitis (for example, fulminant hepatitis, chronic hepatitis, viral hepatitis, alcoholic hepatitis, etc.) ), AIDS, rejection after organ transplantation, septic shock, congestive heart failure, myelodysplastic syndrome, neurodegenerative diseases (e.g. neuropathy due to cerebral ischemia, Chinton disease, there is provided Parkinson's disease, amyotrophic lateral sclerosis, useful peptides and a pharmaceutically acceptable salt thereof in the treatment of such as Alzheimer's, etc.) and cancer.
[0002]
[Prior art]
Apoptosis is a natural death necessary for maintaining the homeostasis of the living body, and its failure leads to serious diseases such as cancer and autoimmune diseases (Annu. Rev. Immunol., 17, 221-253, 1999). Apoptosis is also caused by external factors such as radiation and drugs, but TNF, TRAIL, FasL, etc. have been reported as endogenous inducers (Pharm Acta Helv, 74, 281-286, 2000; Exp Cell Res, 256, 58-66, 2000). These bind to each specific receptor on the target cell and induce apoptosis (TIBS, 24 February, 47-53, 1999).
[0003]
On the other hand, overproduction of TNF, TRAIL, FasL, etc. is caused by rheumatoid arthritis, multiple sclerosis, AIDS (acquired immune deficiency syndrome), cancer, septic shock, congestive heart failure. (Congestive heart failure), Aplastic anemia, Myelodysplastic syndrome, Crohn's disease, and other diseases (Microsc Res Tech, 50, 229-235, 2000 ).
[0004]
A humanized immunoglobulin that specifically reacts with FasL is known, and it has been shown to suppress a physiological reaction between FasL and Fas typified by apoptosis (WO98 / 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 in the Fas / FasL system, is known (Japanese Patent Laid-Open No. 2000-169393).
[0006]
It is known that a protein that binds to Caspase-8 and does not have a Death effector domain has an ability to control apoptosis (Japanese Patent Laid-Open No. 2000-226400).
[0007]
MORT-1 that binds to the intracellular domain of Fas, or a protein that binds to TRADD and modulates the effect of FasL or TNF, as well as a peptide that inhibits the activity are known (Japanese Patent Laid-Open No. 11-509422). ).
[0008]
An inhibitor or preventive agent for apoptosis caused by the interaction between FasL on cytotoxic T cells and Fas on organ cell membrane, which contains epidermal growth factor (EGF) as an active ingredient, is known (Japanese Patent Laid-Open No. Hei 9 (1994)). 10-194888).
[0009]
A synthetic peptide obtained by searching for a TNF-binding functional part of a natural TNF-binding protein is known, and has been shown to inhibit TNF cytotoxicity against mouse LM cells (Japanese Patent Laid-Open No. 5-194594).
[0010]
An apoptosis inhibitor characterized by being a condensed ring compound of a 7-membered ring composed of triazepine or diazepine and a 5-membered ring containing 2 to 4 nitrogen atoms is known (Japanese Patent Laid-Open No. 11-228576).
[0011]
In addition, in recent years, drugs that inhibit apoptosis due to caspase inhibitory action are neuropathy caused by cerebral ischemia (Proc Natl Acad Sci USA, 95, 15769-15774, 1998), Huntington's disease (Nature Medicine, 6, 797-801, 2000), Parkinson's disease (J Neurosci, 22, 1763-1771, 2002), amyotrophic lateral sclerosis (Nature, 417, 74-78, 2002), effective treatment in animal models of neurodegenerative diseases such as Alzheimer It has been reported to show an effect.
[0012]
Apoptosis inhibitors typified by antibodies are proteins with high specificity but large molecular weight, and thus have problems in antigenicity, administration method, stability, and the like. A synthetic peptide obtained by exploring the TNF-binding functional part of a natural TNF-binding protein has problems such as reduced effectiveness due to competition with the natural TNF-binding protein and short residence time in the blood. There is. In addition, low molecular weight organic compound apoptosis inhibitors inhibit the basic pathway of apoptosis, and thus have a side effect due to low selectivity.
[0013]
[Patent Document 1]
JP 2002-138099 A
[0014]
[Problems to be solved by the invention]
It is an object of the present invention to provide a novel substance that inhibits apoptosis or inflammation that overcomes the disadvantages found in known apoptosis inhibitors such as the above-described antibodies, synthetic peptides, and small organic compounds. . That is, an object of the present invention is to provide an inhibitor of apoptosis or inflammation that has high selectivity, good efficacy, and not so high molecular weight.
[0015]
[Means for Solving the Problems]
According to the present invention, the above-described problem is that a peptide containing any of the following amino acid sequences (however, the N-terminal amino group may be acylated and the C-terminal carboxyl group is amidated or esterified. Or a salt thereof having an activity of inhibiting the induction of apoptosis or inflammation:
(A) Any of the following amino acid sequences:
Tyr Arg His Ala Trp Ser Glu Asn Leu Ala Gln Cys Phe Asn (SEQ ID NO: 1);
Phe Arg Glu Glu Asp Ser Pro Glu Thr Cys Arg Lys (SEQ ID NO: 2);
Phe Cys Asn Ser Thr Val Cys Glu His (SEQ ID NO: 3);
This is solved by providing (b) an amino acid sequence in which any amino acid residue not exceeding 6 is deleted, added and / or substituted in any amino acid sequence of (a) above.
[0016]
DETAILED DESCRIPTION OF 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
[0017]
In the present specification, the amino acid sequence of a peptide is described according to a conventional method 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.
[0018]
Production of the peptide of the present invention is carried out by an ordinary 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, “Sequence Chemistry Experiment Course 2 Protein Chemistry (Part 2)” edited by the Japanese Biochemical Society (Showa 62). May 20, 2000, published by Tokyo Chemical Co., Ltd.), pages 641-694].
[0019]
In the preparation of the peptide of the present invention by the solid phase synthesis method, for example, a polymer that is insoluble in a reaction solvent such as a styrene-divinylbenzene copolymer has an amino acid corresponding to the C-terminus of the target peptide. An α-amino other than the α-COOH group, which is bound via the α-COOH group and then has the corresponding amino acid or peptide fragment in the direction of the N-terminus of the target peptide. An operation of protecting and condensing a functional group such as a group, and an operation of removing a protecting group of an amino group forming a peptide bond such as an α-amino group in the bound amino acid or peptide fragment In order to elongate the peptide chain to form a peptide chain corresponding to the target peptide, and then Tide chains were detached from the polymer and obtain a peptide of interest by removing the protecting group from the functional group being protected, and then carried out by purification. 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. Moreover, it is effective to purify the obtained peptide by reverse phase liquid chromatography or gel permeation chromatography.
[0020]
Moreover, the salt of the peptide of this invention is prepared by utilizing normal salt formation reaction.
[0021]
As described above, the amino acid sequence to be included in the peptide of the present invention is as follows.
(1): Tyr Arg His Ala Trp Ser Glu Asn Leu Ala Gln Cys Phe Asn (SEQ ID NO: 1)
(2) Phe Arg Glu Glu Asp Ser Pro Glu Thr Cys Arg Lys (SEQ ID NO: 2),
(3) Phe Cys Asn Ser Thr Val Cys Glu His (SEQ ID NO: 3),
The thing selected from is mentioned.
Specific examples of amino acid sequences included in (b) above include (4) Gln Cys Glu Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro Glu Thr Cys Arg Lys (SEQ ID NO: 4), (5) Phe Arg Glu Glu Asp Ser Pro Glu Thr Cys Arg Lys Cys Arg Thr (SEQ ID NO: 5), (6) Phe Cys Glu Glu Asp Ser Pro Glu Thr Cys Arg Lys (SEQ ID NO: 6), (7) Phe Ser Asn Ser Thr Val Ser Glu His (sequence number: 7) etc. are mentioned.
[0022]
That is, the peptide of the present invention includes a peptide having the amino acid sequence of any one of SEQ ID NOs: 1 to 3 as all or part of the sequence. In some cases, the amino group of the N-terminal amino acid residue of the amino acid sequence may be acylated. For example, the amino group may have a lower alkanoyl group (for example, an acetyl group) or a lower alkoxycarbonyl group (for example, a methoxycarbonyl group). Alternatively, a butoxycarbonyl group or the like may be introduced. The carboxyl group of the C-terminal amino acid residue of the amino acid sequence may be amidated or esterified. For example, the carboxyl group is an amide group or a lower alkyl ester group (for example, a methyl ester group or a butyl ester group). It may be converted to. The amino acid sequence described in any one of SEQ ID NOs: 1 to 3 contained in the peptide of the present invention may be one in which each amino acid residue is homologously substituted based on the properties of the side chain functional group. For example, Cys can be replaced by Ser, Leu can be replaced by Ile and Val, Ser can be replaced by Thr, Glu can be replaced by Asp, Gln can be replaced by Asn, Lys can be replaced by Arg, and Phe can be replaced by Tyr, His, and Trp. Can also be replaced. Furthermore, at any position in the amino acid sequence of any one of SEQ ID NOs: 1 to 3 contained in the peptide of the present invention, 1 to 6 arbitrary amino acid residues are preferably 1 or 2 And more preferably one arbitrary amino acid residue may be independently added (for example, SEQ ID NO: 4 or 5 corresponding to SEQ ID NO: 2). 6 arbitrary amino acid residues, preferably 1 to 3 arbitrary amino acid residues, more preferably 1 or 2 arbitrary amino acid residues, still more preferably 1 arbitrary amino acid residue May be independently deleted. In the present specification, the description of “the peptide of the present invention” includes all of these peptides.
[0023]
The peptides of the present invention may form salts, in particular pharmaceutically acceptable salts, forming acid salts, metal salts or amine salts. Examples of the salt include, but are not limited to, hydrochloride, sulfate, phosphate, lactate, tartrate, maleate, fumarate, oxalate, malate, citrate, oleic acid Salts, palmitates, etc .; alkali metal salts (eg, sodium, potassium, etc.) or alkaline earth metal salts (eg, calcium, etc.), or aluminum salts (such as salts with hydroxide ions or carbonate ions) And triethylamine salt, benzylamine salt, diethanolamine salt, t-butylamine salt, dicyclohexylamine salt, arginine salt and the like.
[0024]
The inhibitory effect of the peptide of the present invention on TNF, TRAIL, FasL and the like is performed using a usual method for measuring these activities. For example, the activity of TNF is performed by measuring (1) cytotoxic activity targeting mouse L cells, (2) inhibitory activity of lipoprotein lipase, and (3) proliferation promoting activity against fibroblasts [for example, Nakajima Atsushi et al., New Basic Biochemical Experimental Method 6 “Measurement method using biological activity” (published on Mar. 30, 1988, Maruzen Co., Ltd., pp. 267-272). TNF activity can be measured by inducing apoptosis of mouse L929 cells or NIH3T3 cells. The activity of TRAIL can be measured by inducing apoptosis in mouse L929 cells. FasL activity can be measured by inducing apoptosis in mouse L929 cells or NIH3T3 cells into which Fas gene has been introduced (see, for example, EMBO J, 13, 4587-4596, 1994). For the detection of apoptosis induction, a method for detecting a change in a 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. Of these, propidium iodide (PI) and FITC-labeled annexin V are particularly preferable.
[0025]
The treatment method using the peptide of the present invention includes, for example, systemic administration such as intravenous administration, subcutaneous administration, intraperitoneal administration, transdermal administration, oral administration, etc. And a method for injecting the peptide of the present invention.
[0026]
The dosage for effective activity expression of the peptide of the present invention is usually 0.01 μg / kg to 2 g / kg (body weight), preferably 0.01 μg / kg to 200 mg / kg (body weight). It is.
[0027]
The administration form is preferably a solution obtained by dissolving the peptide in a pharmaceutically acceptable solution such as 5% glucose solution or physiological saline, and the solution contains various pharmacologically acceptable additives. You may go out. It is also possible to encapsulate or liposome peptides.
[0028]
Antibody production
Various host animals can be immunized by injecting a composition comprising one or more peptides of the invention. 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, such as Freund (complete or incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin , Pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol and potentially useful human adjuvants such as BCG (sterilized bovine tuberculosis) and Corynebacterium parvum However, it is not limited to these.
[0029]
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 are included.
[0030]
Monoclonal antibodies, which are homogeneous collections of antibodies against specific antigens, use the peptides described above, and standard hybridoma technology (Kohler et al .: Nature, 256: 495, 1975; Kohler et al .: Eur. J. Immunol., 6: 511, 1976; Kohler et al .: Eur. J. Immunol., 6: 292, 1976; Hammering et al .: In: Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981; US Patent 4,376,110; Cosboa et al .: Immunology Today, 4 Cole et al: Proc. Natl. Acad. Sci. USA, 80: 2026, 1983; Cole et al: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1983) By adopting, it can be prepared. Such antibodies can be selected from any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD and their subclasses. Hybridomas producing monoclonal antibodies may be cultured either in vitro or in vivo.
[0031]
Antibodies can be used, for example, to detect the presence of an antigen in a biological sample as part of a diagnostic assay, and to be used for evaluation of medical treatment effects by other therapeutic methods. You can also.
[0032]
Immunoassay
Further embodiments of the invention relate to the field of immunoassays using antibodies that specifically recognize one or more peptides of the invention as antigens.
[0033]
One or more peptides of the present invention are specifically recognized as the antigen.
[0034]
In one embodiment, the immunoassay is an enzyme linked immunoassay (ELISA). In this type, the sample and a given reagent are contained within the wells of a microtiter well plate and the assay is initiated by adding a detection reagent appropriate to the detection of the enzyme label to the well. Such assays are known to those skilled in the art, and background information relating to the techniques of such immunoassays can be found in 'The Immunoassay Handbook', (Wild, DG Ed, Stockton Press, New York, [1994]). it can.
[0035]
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 is a particularly preferred enzyme label for use in the enzyme immunoassay method of the present invention.
[0036]
In yet another embodiment, the immunoassay is a radioimmunoassay, and radioisotopes suitable for use in the assay of the present invention emit β-emitting isotopes such as tritium and Auger electrons. Iodine-125 is included.
[0037]
In yet another embodiment, the immunoassay is a fluorescent immunoassay and the appropriate fluorescent label is selected from fluorescein, rhodamine and cyanine dyes.
[0038]
Furthermore, as another embodiment of the present invention, there is provided a nucleic acid encoding the peptide of the present invention in addition to the peptide of the present invention.
[0039]
Example
Hereinafter, the present invention will be described specifically by way of examples. In addition, this invention is not limited by these Examples.
(Example 1)
Formula (1): Tyr Arg His Ala Trp Ser Glu Asn Leu Ala Gln Cys Phe Asn (SEQ ID NO: 1) peptide containing the amino acid sequence Tyr Arg His Ala Trp Ser Glu Asn Leu Ala Gln Cys Phe Asn-NH ₂ Was synthesized by a solid phase synthesis method using an automatic peptide synthesizer. That is, styrene-divinylbenzene copolymer having 4- (2 ′, 4′-dimethoxyphenyl-fluorenylmethoxycarbonyl) -aminomethyl) -phenoxyacetamido-ethyl group at a rate of 0.62 mmol / g (resin) Using 0.1 mmol of a granular resin (Fmoc amide resin, manufactured by Applied Biosystems, USA) composed of a polymer (constituent molar ratio of styrene and divinylbenzene: 99 to 1) from the carboxyl terminal to the amino terminal of the target peptide. The corresponding amino acids were combined sequentially. In the binding reaction, amino acid N, manufactured by Applied Biosystems, USA ^α -9- (Fluorenylmethoxycarbonyl) -N ^γ -Trityl-L-glutamine [Fmoc glutamine], N ^α -9- (fluorenylmethoxycarbonyl) -γ-butyl-L-glutamic acid [Fmoc glutamic acid], N ^α -9- (Fluorenylmethoxycarbonyl) -N ^β -Trityl-L-asparagine [Fmoc asparagine], N ^α -9- (Fluorenylmethoxycarbonyl) -N ^G -(2,2,5,7,8-pentamethylchroman-6-sulfonyl) -L-arginine [Fmoc arginine], N ^α -9- (Fluorenylmethoxycarbonyl) -S-trityl-L-cysteine [Fmoc cysteine], N ^α -9- (fluorenylmethoxycarbonyl) -Ot-butyl-L-serine [Fmoc serine], N ^α -9- (fluorenylmethoxycarbonyl) -L-leucine [Fmoc leucine], N ^α -9- (Fluorenylmethoxycarbonyl) -N ^Im -Trityl-L-histidine [Fmoc histidine], N ^α -9- (fluorenylmethoxycarbonyl) -L-phenylalanine [Fmoc phenylalanine], N ^α -9- (Fluorenylmethoxycarbonyl) -L-alanine [Fmocalanine], N ^α -9- (fluorenylmethoxycarbonyl) -Ot-butyl-L-tyrosine [Fmoc tyrosine], N ^α -9- (Fluorenylmethoxycarbonyl) -N ^Ind -Butoxy-L-tryptophan [Fmoc tryptophan] was used in an amount of 1 mmol for each binding step.
[0040]
The obtained peptide resin was treated with 10 ml of trifluoroacetic acid containing 7.5% phenol, 2.5% ethanedithiol, 5% water and 5% thioanisole for 3 hours. The resulting solution was added to diethyl ether, and the resulting precipitate was further washed several times with diethyl ether to deprotect the peptide and desorb from the resin. The crude product was purified with a PD10 column (Amersham Pharmacia Japan) to obtain a peptide. AKTA explorer 10XT manufactured by Pharmacia Biotech Co., Ltd. [column: Novapack C18 manufactured by Millipore Waters Co., Ltd., 3.9 mmφ × 150 mm, mobile phase: a mixed solvent of acetonitrile and water containing 0.05% by volume of trifluoroacetic acid ( Acetonitrile concentration was changed linearly from 5 vol% to 50 vol% over 30 minutes) and a flow rate of 1.0 ml / min] showed a single peak at 17.3 min. The molecular weight of the purified peptide determined by FAB method mass spectrum was 1738 (theoretical value: 1737.92).
[0041]
(Example 2)
Formula (2): Phe Arg Glu Glu Asp Ser Pro Glu Thr Cys Arg Lys (SEQ ID NO: 2), Formula (3): Phe Cys Asn Ser Thr Val Cys Glu His (SEQ ID NO: 3), Formula (4): Gln Cys Glu Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro Glu Thr Cys Arg Lys (SEQ ID NO: 4), Formula (5): Phe Arg Glu Glu Asp Ser Pro Glu Thr Cys Arg Lys Cys Arg Thr (SEQ ID NO: 5 ), Formula (6): Phe Cys Glu Glu Asp Ser Pro Glu Thr Cys Arg Lys (SEQ ID NO: 6), Formula (7): Phe Ser Asn Ser Thr Val Ser Glu His (SEQ ID NO: 7) A peptide containing the sequence was synthesized in the same manner as in Example 1. However, in the binding reaction, in addition to the amino acids shown in Example 1, N, manufactured by Applied Biosystems, USA ^α -9- (fluorenylmethoxycarbonyl) -glycine [Fmoc glycine], N ^α -9- (fluorenylmethoxycarbonyl) -proline [Fmoc proline], N ^α -9- (fluorenylmethoxycarbonyl) -Ot-butyl-L-threonine [Fmoc threonine], N ^α -9- (Fluorenylmethoxycarbonyl) -N ^ε -T-butoxycarbonyl-L-lysine [Fmoc lysine], N ^α -9- (fluorenylmethoxycarbonyl) -L-valine [Fmoc valine], N ^α -9- (Fluorenylmethoxycarbonyl) -β-butyl-L-aspartic acid [Fmoc aspartic acid] was used in an amount of 1 mmol for each coupling step.
[0042]
Phe Arg Glu Glu Asp Ser Pro Glu Thr Cys Arg Lys-NH ₂ In the purified peptide represented by (1), a single peak was exhibited at 10.0 min, and the molecular weight of the purified peptide was 1496 (theoretical value: 1495.64). Phe Cys Asn Ser Thr Val Cys Glu His-NH ₂ In the purified peptide represented by (1), a single peak was shown at 10.2 min, and the molecular weight of the purified peptide was 1039 (theoretical value: 1038.17). Gln Cys Glu Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro Glu Thr Cys Arg Lys-NH ₂ In the purified peptide represented by (1), a single peak was shown at 10.9 min, and the molecular weight of the purified peptide was 2144 (theoretical value: 2143.30). Phe Arg Glu Glu Asp Ser Pro Glu Thr Cys Arg Lys Cys Arg Thr-NH ₂ In the purified peptide represented by (1), a single peak was shown at 9.8 min, and the molecular weight of the purified peptide was 1856 (theoretical value: 1856.07). Phe Cys Glu Glu Asp Ser Pro Glu Thr Cys Arg Lys-NH ₂ In the purified peptide represented by (1), a single peak was shown at 9.0 min, and the molecular weight of the purified peptide was 1443 (theoretical value: 1442.59). Phe Ser Asn Ser Thr Val Ser Glu His-NH ₂ In the purified peptide represented by (1), a single peak was shown at 8.3 min, and the molecular weight of the purified peptide was 1006 (theoretical value: 1006.04).
[0043]
(Test Example 1)
Dulbecco containing 50,000 L929 cells or 10% fetal calf serum dispersed in Eagle's medium (10% FCS / E-MEM) containing 10% fetal calf serum in each well of a 24-well plate (NUNC) 1 ml of 50,000 NIH3T3 cells dispersed in modified Eagle medium (10% FCS / D-MEM) were dispensed. 37 ° C, 5% CO ₂ After 24 hours of culturing to allow the cells to adhere, 100 μl of a phosphate buffer (PBS: 10 mM, containing 0.15 M sodium chloride, pH 7.4) solution of the peptide of the present invention, and rhTNFα (R & D Systems) ) Or rhTRAIL (R & D Systems) or rhFasL (R & D Systems) in PBS (10 μl) was added to each well. When using rhFasL (R & D Systems) or rhTRAIL, 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, and further 37 ° C., 5% CO 2. ₂ Let stand for 15 minutes. The fluorescence of PI was observed with a confocal laser microscope to discriminate cells induced by apoptosis. The ratio of cells induced by apoptosis and total cells in five arbitrarily selected visual fields was calculated and used as the apoptosis induction rate.
[0044]
When rhTNFα was used at 50 ng / ml for L929 cells and PBS was added instead of the peptide solution, the apoptosis induction rate was 61%. Tyr Arg His Ala Trp Ser Glu Asn Leu Ala Gln Cys Phe Asn-NH ₂ Was added to a final concentration of 100 μg / ml, the apoptosis induction rate was 43% and the apoptosis inhibition rate was 29%.
[0045]
Similarly, the apoptosis induction rate was 46% when rhTRAIL was used at 50 ng / ml for L929 cells and PBS was added instead of the peptide solution. Phe Arg Glu Glu Asp Ser Pro Glu Thr Cys Arg Lys-NH ₂ When the peptide was added to a final concentration of 400 μg / ml, the apoptosis inhibition rate was 59%, and Gln Cys Glu Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro Glu Thr Cys Arg Lys-NH ₂ When the peptide was added to a final concentration of 400 μg / ml, the inhibition rate of apoptosis was 47%, Phe Arg Glu Glu Asp Ser Pro Glu Thr Cys Arg Lys Cys Arg Thr-NH ₂ When the final peptide concentration of 400 μg / ml was added, the inhibition rate of apoptosis was 44%, Phe Cys Glu Glu Asp Ser Pro Glu Thr Cys Arg Lys-NH ₂ When the peptide was added to a final concentration of 400 μg / ml, the apoptosis inhibition rate was 61%.
[0046]
When rhFasL was used at 50 ng / ml for NIH3T3 cells and PBS was added instead of the peptide solution, the apoptosis induction rate was 61%. Phe Cys Asn Ser Thr Val Cys Glu His -NH ₂ When the final peptide concentration of 200 μg / ml was added, the apoptosis inhibition rate was 21%, Phe Ser Asn Ser Thr Val Ser Glu His-NH ₂ When the peptide was added to a final concentration of 200 μg / ml, the apoptosis inhibition rate was 19%.
[0047]
【The invention's effect】
According to the present invention, a novel peptide or a salt thereof that inhibits new apoptosis or inflammation is provided, which overcomes the disadvantages found in conventionally known apoptosis inhibitors such as antibodies, synthetic peptides and small organic compounds. That is, a novel peptide that inhibits apoptosis or inflammation with high selectivity, good efficacy, and low molecular weight is provided.
Further, as is clear from the above examples and test examples, the novel peptide provided by the present invention or a pharmaceutically acceptable salt thereof can effectively inhibit apoptosis induction by TNF, TRAIL, FasL, etc. So autoimmune diseases caused by these (e.g. systemic lupus erythematosus, Hashimoto's disease, rheumatoid arthritis, graft-versus-host disease, Sjogren's syndrome, pernicious anemia, Addison's disease, scleroderma, Goodpascher's syndrome, Crohn's disease, Autoimmune hemolytic anemia, spontaneous infertility, myasthenia gravis, multiple sclerosis, Based's disease, idiopathic thrombocytopenic purpura, insulin-dependent diabetes), allergy, atopy, arteriosclerosis, myocarditis, Cardiomyopathy, glomerulonephritis, aplastic anemia, hepatitis (eg, fulminant hepatitis, chronic hepatitis, viral hepatitis, alcoholic hepatitis, etc.), AIDS, organ transplantation Rejection, septic shock, congestive heart failure, spinal dysplasia syndrome, neurodegenerative diseases (e.g. neuropathy due to cerebral ischemia, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, Alzheimer, etc.) and cancer It is useful as a treatment agent.
[0048]
[Sequence Listing]

Claims (12)

A peptide consisting of any of the following amino acid sequences (however, the amino group at the N-terminus may be acylated and the carboxyl group at the C-terminus may be amidated or esterified) or a salt thereof: Having activity of inhibiting the induction of apoptosis or inflammation:
Tyr Arg His Ala Trp Ser Glu Asn Leu Ala Gln Cys Phe Asn (SEQ ID NO: 1);
Phe Arg Glu Glu Asp Ser Pro Glu Thr Cys Arg Lys (SEQ ID NO: 2);
Phe Cys Asn Ser Thr Val Cys Glu His (SEQ ID NO: 3);
Gln Cys Glu Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro Glu Thr Cys Arg Lys (SEQ ID NO: 4);
Phe Arg Glu Glu Asp Ser Pro Glu Thr Cys Arg Lys Cys Arg Thr (SEQ ID NO: 5);
Phe Cys Glu Glu Asp Ser Pro Glu Thr Cys Arg Lys (SEQ ID NO: 6); and
Phe Ser Asn Ser Thr Val Ser Glu His (SEQ ID NO: 7).   The peptide or a salt thereof according to claim 1, wherein a lower alkanoyl group or a lower alkoxycarbonyl group is introduced by acylation of the N-terminal amino group.   The peptide or a salt thereof according to claim 1, wherein an amide group or a lower alkyl ester group is introduced by amidation or esterification of a C-terminal carboxy group.   The salt of the peptide in any one of Claims 1-3 whose salt is an acid salt, a metal salt, or an amine salt.   The acid salt is hydrochloride, sulfate, phosphate, lactate, tartrate, maleate, fumarate, oxalate, malate, citrate, oleate or palmitate. 5. A salt of the peptide according to 4.   The peptide salt according to claim 4, wherein the metal salt is an alkali metal salt, an alkaline earth metal salt or an aluminum salt.   The peptide salt according to claim 4, wherein the amine salt is a triethylamine salt, a benzylamine salt, a diethanolamine salt, a t-butylamine salt, a dicyclohexylamine salt or an arginine salt.   The peptide or a salt thereof according to any one of claims 1 to 7, wherein the induction of apoptosis or inflammation is caused by TNF.   The peptide or a salt thereof according to any one of claims 1 to 7, wherein the induction of apoptosis or inflammation is caused by TRAIL.   The peptide or a salt thereof according to any one of claims 1 to 7, wherein the induction of apoptosis or inflammation is caused by FasL.   Autoimmune disease, allergy, atopy, arteriosclerosis, myocarditis, cardiomyopathy, glomerulonephritis, aplastic anemia, hepatitis, AIDS, rejection after organ transplantation, septic shock, congestive heart failure, myelodysplastic syndrome, The peptide or a salt thereof according to any one of claims 1 to 10, which is useful for treatment of a neurodegenerative disease or cancer.   A nucleic acid encoding the peptide of claim 1.