JP2010120937A - Inflammatory disease preventing and treating agent containing amino acid fragment of human apolipoprotein a-ii or its modification - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a side effect-reduced preventing and treating agent for inflammatory diseases such as hepatitis accompanying the production of inflammatory cytokines, the production of chemokines or the growth of T-cells and rheumatism. <P>SOLUTION: It has been determined that a peptide which is present in the region of Sequence No.2 of a fragment of apolipoprotein A-II and is composed of 8-30 amino acids including at least the amino acid sequence of Sequence No.23 or the amino acid sequence of Sequence Nos.4 and 5 or a modification thereof inhibits the production of inflammatory cytokines and chemokines or inhibits the growth of T-cells and a drug containing the peptide or the modification can prevent and treat inflammatory diseases. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention is a peptide that suppresses the production of inflammatory cytokines, suppresses the production of chemokines or suppresses the proliferation of T cells, or a variant thereof, and an agent for preventing or treating inflammatory diseases. Furthermore, the present invention is a peptide having an anti-inflammatory action site in the amino acid sequence of human apolipoprotein A-II or a variant thereof, and an inflammatory disease preventing / treating agent containing the peptide.

  It is the immunocompetent cells that play an important role in the production of inflammatory cytokines, chemokines or T cell proliferation. Lymphocytes (T cells, B cells, monocytes, neutrophils, etc.) present in the blood and bone marrow, etc., and cells resident in various organs such as the brain and liver (macrophages, dendritic cells, vascular endothelial cells) All cells involved in the immune response, such as tissue parenchymal cells) are immunocompetent cells, and these cells secrete cytokines, chemokines, and various inflammatory substances as humoral factors that activate the immune response . In addition to humoral factors, cellular immunity that exerts an effect by cell-cell interaction is also activated.

  Inflammation, which is a direct phenomenon of immune system activation, is a biological defense reaction in which the immune system works when body tissues are damaged by bacterial infection, chemical action, or physical action, thereby causing swelling, pain, fever, etc. It is a symptom that appeared in the living body. In such an inflammatory site, various inflammatory cytokines and chemokines are produced, and many lymphocytes infiltrate the inflammatory site. The main inflammatory cytokine is TNF-α, but IFN-γ and MCP-1 are also well known. In addition, when lymphocytes are activated by an antigen, type 1 helper T cells (Th1 cells) in the lymphocytes are cytokines such as interleukin 2 (IL-2) and interferon-γ (IFN-γ). It is known that lymphocytes, especially T cells, proliferate and differentiate by IL-2 and IFN-γ produced.

  MCP-1 (monocyte chemotactic protein-1) is also called monocyte chemotactic and activating factor (MCAF), and constitutes a family with IL-8 due to its structural and functional similarities. It has monocyte-specific migration activity and eosinophil histamine releasing action, and is thought to be deeply involved in inflammation and biological defense. In particular, it is assumed that monocyte chemotaxis and activation are involved in various inflammations, autoimmune diseases, arteriosclerosis and tumors, and basophil activation is involved in allergic reactions. Yes.

  MCP-1 acts on monocytes and macrophages to play a role in promoting chemotaxis enhancement and activation in the local area of inflammation, and is thought to be involved in the development of several inflammatory reactions. Therefore, it is considered that a substance that suppresses the action of MCP-1 is useful for the prevention or treatment of these inflammatory reactions. Monoclonal antibody against MCP-1 (Patent Document 1), antibody fragment (Patent Document 2), receptor protein (Patent Document) 3) has been reported. However, substances that suppress the action of MCP-1 have not been used clinically.

  TNF-α is mainly produced from macrophages, and is also produced from neutrophils, fibroblasts, lymphocytes (T cells), NK cells, mast cells, etc., and damages tumor cells (antitumor effect). It is also an intrinsic pyrogen. A low concentration of TNF-α acts on vascular endothelial cells, enhances the expression of adhesion molecules, and promotes the production of cytokines such as IL-1 and IL-6. A high concentration of TNF-α promotes the production of acute phase proteins and suppresses IFN-α production in the liver.

  If TNF-α continues to be produced excessively for some reason, it causes various diseases such as rheumatoid arthritis, ulcerative colitis, Crohn's disease, and type 2 diabetes. Therefore, drugs that suppress the production of inflammatory cytokines such as TNF-α have been developed for patients suffering from such diseases. In addition to existing anti-inflammatory agents such as ibuprofen and indomethacin, thalidomide derivatives (non-patent literature) 1), pyrazolone derivatives (see Non-Patent Document 2), alkaloids of the genus Euphorbiaceae (see Patent Document 4), proteoglycans (see Patent Document 5), HDL, apolipoprotein AI and apolipoprotein A-II (patent literature) 6)) has been reported.

  Recently, TNF-α inhibitors have been developed to cure inflammatory diseases such as rheumatoid arthritis, ulcerative colitis, Crohn's disease, and type 2 diabetes by suppressing the action of inflammatory cytokines, and contributed greatly to treatment. ing. However, on the other hand, in the TNF-α monoclonal antibody preparation, anaphylaxis-like symptoms (fever, dyspnea, bronchospasm, hypotension, angioedema, cyanosis, urticaria) occurred after infusion, and 3 days or more passed after administration. Severe delayed-acting hypersensitivity (muscle pain, rash, fever, pruritus, edema, urticaria) may occur later, and as an important side effect, it strongly suppresses immunity, causing infection (sepsis , Opportunistic infections including fungal infections).

IL-2 is a cytokine secreted mainly by T cells (CD4 +, CD8 +) and consists of 133 amino acids. IL-2, also called T cell growth factor (TCGF), proliferates T cells, but IL-2 affects not only T cells but also B cells, NK cells, macrophages, neutrophils, etc. It has been known. The IL-2 receptor is expressed on the cell surface of these cells and is an important regulator of immune and inflammatory responses.
IL-2 production suppression is expected to show therapeutic effects in disease states such as rheumatoid arthritis, atopic dermatitis, psoriasis, and tissue graft rejection.

  IFN-γ is produced by activated T cells and has a regulatory effect on the immune system and inflammatory response, and is detected in tissues and blood in many diseases. Diseases that IFN-γ is considered to be strongly involved in pathogenesis include viral infections, infections caused by microorganisms other than viruses, rheumatoid arthritis, collagen diseases such as SLE, arteriosclerosis, diabetes, fulminant hepatitis, Malignant tumor, Kawasaki disease, etc.

  T cells are a type of lymphocytes, and progenitor cells produced in the bone marrow are differentiated and matured through selection in the thymus. Normal T cells are an integral part of a mammal's immune response, but pathological conditions may develop due to abnormal proliferation of T cells. For example, autoimmune diseases include systemic lupus lupus erythematosus, rheumatoid arthritis and multiple sclerosis. T cells are an integral part of the immune response. Therefore, treatments aimed at inhibiting T cell proliferation are highly desirable for treating autoimmune diseases.

  Human apolipoprotein A-II is a major component of high-density lipoprotein (HDL) that is resident in human blood, and its chemical structure has already been clarified. A polypeptide consisting of amino acids. This human apolipoprotein A-II is known to exist as a dimer disulfide-bonded at the cysteine of the sixth residue in HDL (Non-patent Document 3).

  As active sites of human apolipoprotein A-II, peptides having the amino acid sequence from the 17th to the 30th (17-30) and the amino acid sequence from the 51st to the 60th (51-60) from the N-terminal are known. (Patent Document 7). However, there is no specific description of other active sites, and there is no description of amino acid sequences that suppress anti-inflammatory or cytokines. Moreover, about Usia polypoprotein A-II, it describes about the antibacterial activity (refer patent document 8). Although Usia polypoprotein A-II, Usia polypoprotein A-II fragment and human apolipoprotein A-II are described to show fibroblast proliferating activity (see Patent Document 9), they suppress anti-inflammatory or cytokines There is no description about the amino acid sequence to be performed.

Examples of peptides that suppress inflammation include apolipoprotein AI fragment (see Patent Document 6), peptides that suppress cytokines such as TNF and IL-6 (Patent Document 10), and chemokine receptor CCR3 antagonist polypeptide (Patent Document 11) ApoE receptor binding peptide (Patent Document 12) and the like are known, but there is no knowledge of apolipoprotein A-II fragments.
Japanese Patent No. 3920215 WO 2004/024921 JP-A-9-238688 JP 2003-26586 A JP 2007-131548 A Special table 2004-500105 gazette WO87 / 02062 Publication Japanese Patent No. 3733485 Japanese Patent No. 3913537 JP 2004-196707 A JP 2001-29079 A JP-T-2005-511514 S. Niwayama et al. , J. et al. Med. Chem. , 39, 3044-3045, 1996 M.M. P. Clark et al. , J. et al. Med. Chem. , 47, 2724-2727, 2004 The Lipid Vol. 2, no. 3 243-249, 1991

  An object of the present invention is to provide an agent for treating or preventing an inflammatory disease having no side effect, including a peptide having an anti-inflammatory site in the amino acid sequence of human apolipoprotein A-II.

As a result of preparing various fragment peptides from the amino acid sequence of human apolipoprotein A-II (SEQ ID NO: 1) consisting of 77 amino acids and examining the various physiological characteristics of each fragment, The present inventors have found that the peptide fragment has anti-inflammatory properties and completed the present invention.
That is, the present invention
(1) A prophylactic / therapeutic agent for inflammatory diseases comprising a peptide consisting of 8 to 30 consecutive amino acids or a variant thereof in the peptide of SEQ ID NO: 2.
(2) A peptide consisting of 8 to 30 consecutive amino acids or a variant thereof suppresses production of MCP-1, TNF-α, IFN-γ or IL-2, or suppresses T cell proliferation. (1) The preventive / therapeutic agent for inflammatory diseases according to the above.
(3) The prophylactic / therapeutic agent for inflammatory diseases according to (1) or (2), wherein the peptide comprising 8 to 30 consecutive amino acids comprises the peptide of SEQ ID NO: 23.
(4) The prophylactic / therapeutic agent for inflammatory diseases according to (1) or (2), comprising 20 to 30 consecutive amino acids including the peptide of SEQ ID NO: 4 or 5 in the peptide of SEQ ID NO: 2.
(5) Peptide variants are generated by substitution of one or more amino acids, deletion of one or more amino acids, addition of one or more amino acids, or linkage of SS bonds (1 ) The inflammatory disease preventing / treating agent according to any one of (4) to (4).
(6) The prophylactic / therapeutic agent for inflammatory diseases according to (1) to (4), wherein the modified peptide is produced by N-terminal acetylation or pyrolation or C-terminal amidation.
(7) The prophylactic / therapeutic agent for inflammatory diseases according to (5), wherein the substitution of one or more amino acids is asparagine or serine substituted with alanine.
(8) The inflammatory disease preventive / therapeutic agent according to (5), wherein the substitution of one or more amino acids is generated by substitution of L-form amino acids with D-form amino acids.
(9) The inflammatory disease preventive / therapeutic agent according to (5), wherein the addition of one or more amino acids is performed with a polar amino acid. (10) The addition of a polar amino acid is performed via a spacer amino acid. A prophylactic / therapeutic agent for inflammatory diseases according to (9).
(11) The inflammatory disease preventive / therapeutic agent according to (1), wherein the inflammatory disease is accompanied by an increase in inflammatory cytokines, chemokines or T cells during inflammation.
(12) Diseases accompanied by an increase in inflammatory cytokines, chemokines or T cells are systemic inflammatory response syndrome (SIRS), systemic vasculitis, bacterial osteomyelitis, dermatitis, psoriasis, diabetes, hepatitis, rheumatism, infection Or neuropathy associated with autoimmune disease, neuropathic pain, ischemia reperfusion injury, encephalitis, Guillain-Barre syndrome, Parkinson's disease, arteriosclerosis, metabolic syndrome (metabolic syndrome), heart failure, cardiomyopathy, myocardial infarction, brain The prophylactic / therapeutic agent for inflammatory diseases according to (11), which is a disease comprising infarction, Crohn's disease, graft body disease or transplant rejection.
It is.

  Each peptide described above can be prepared by a peptide synthesis method known as “solid phase method” or “liquid phase method”. For example, “Biochemistry Experiment Course” edited by Japan Biochemical Society, Volume 1, “Protein IV”, pp. 207-495, published by Tokyo Chemical Doujin and edited by “Japan Biochemistry Society” , Volume 1, “Protein VI”, pp. 3-74, 1992, published by Tokyo Chemical Dojin, etc., details of peptide synthesis are described. Moreover, it can synthesize | combine with a peptide synthesizer using Fmoc (9-fluorenyl methoxycarbonyl) solid-phase synthesis method. That is, the Fmoc amino acid introduced with an amino acid corresponding to the C-terminus of each peptide to be synthesized is bound to the resin, and the operations of (I) deprotection and washing of the Fmoc group, and (II) condensation and washing of the Fmoc amino acid are performed. It is possible to synthesize the target peptide by repeatedly extending the peptide chain and finally subjecting it to final deprotection.

  The peptides of the present invention are not limited to those prepared by chemical synthesis. For example, when producing the peptides described in (1) to (13) above, an origin, promoter, ribosome that can replicate in cells An expression vector in which a polynucleotide encoding the peptide is recombinantly prepared may be used as an expression vector having a binding site, a DNA cloning site, a terminator, and the like. After transforming host cells with this expression vector, the obtained transformant may be cultured, and the peptide of this invention may be collected by isolating and purifying the desired peptide from this culture. As a host, prokaryotic cells such as Escherichia coli and Bacillus subtilis and eukaryotic cells such as yeast, insect cells and mammalian cells may be appropriately used.

  In order to isolate and purify the peptide of the present invention, known separation operations can be combined. For example, peptides or proteins such as ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography, high performance liquid chromatography are purified. Methods may be used, and these methods may be appropriately combined as necessary. Then, the purified peptide may be concentrated or lyophilized as necessary to make it liquid or solid depending on the final use form.

Furthermore, in the peptides of the present invention, amino acid substitutions, deletions, or additions can usually be performed according to the common technical knowledge of those skilled in the art. That is, modifications can be made based on known similarities in physical properties such as charge density, hydrophobicity, hydrophilicity, size and shape.

  For example, when substituting individual amino acids, Leu can be replaced with Ser, or vice versa, Ser can be replaced with Leu, and Thr can be replaced with Ser (or vice versa).

  In addition, the charge at both ends of the peptide can be canceled and stabilized by modification such as amidation at the C-terminus of the peptide or acetylation at the N-terminus. In addition, one or more hydrophilic amino acids such as arginine and lysine are added directly to the peptide or added to the peptide via a spacer amino acid in order to improve the membrane permeability of the target cells and the solubility of the peptide. It can also be added. As the amino acid serving as the spacer, proline, glycine and the like are used.

  In some cases, the peptide may be useful for improving the stability and activity of the peptide by cyclization or dimerization using an SS bond between cysteine residues. In addition, when glutamine is at the N-terminal of the peptide, it may be converted to pyroglutamic acid by intramolecular condensation of a carboxyl group and an amino group. In this case, pyroglutamic acid can be added instead of glutamine. .

The peptide of the present invention can be administered orally or parenterally as it is or as a pharmaceutical composition mixed with a known pharmaceutically acceptable carrier or excipient.
Examples of drug forms containing the peptide of the present invention include powders, fine granules, granules, pills, tablets, capsules, troches, syrups, emulsions, ointments, plasters, poultices, suppositories, Examples thereof include eye drops, nasal drops, sprays, injections and the like.

  The dose of the peptide of the present invention varies depending on the age of the patient and the degree of the disease. Generally, in the case of oral administration, the dose is 0.1 μg / kg to 500 mg / kg body weight, preferably 1 μg / kg to 200 mg / day. kg body weight, more preferably 5 μg / kg to 100 mg / kg body weight, 0.1 μg / kg to 500 mg / kg body weight per day for intravenous, intramuscular or subcutaneous administration, preferably 0.5 μg / kg to 200 mg / kg kg body weight, more preferably 1 μg / kg to 100 mg / kg body weight. The number of administration can be divided into 1 to 3 times a day for oral administration and 1 to 2 times a day for injection.

  The peptide of the present invention can be formulated according to a usual method. For example, excipients, excipients, diluents, flavoring agents, fragrances, sweeteners, preservatives, stabilizers, binders, pH adjusters, buffers, surfactants, bases, solvents, fillers, Bulking agent, solubilizer, solubilizer, tonicity agent, emulsifier, suspending agent, dispersant, thickener, gelling agent, adhesive, elastic agent, plasticizer, disintegrant, propellant, storage It can be manufactured as a preparation using a combination of an agent, an antioxidant, a light-shielding agent, a moisturizing agent, an antistatic agent, a soothing agent and the like.

  The administration route of the peptide of the present invention is not particularly limited to oral and parenteral, but parenteral administration such as intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration such as transpulmonary and nasal administration is preferable.

When the prophylactic / therapeutic agent for inflammatory diseases according to the present invention is produced as an injection, according to a conventional method, for example, water for injection, physiological saline, or PBS (0.1 M Add acid buffered saline solution) to dissolve or uniformly suspend, or add pharmacologically acceptable natural / synthetic surfactants (eg lecithins, polysorbates, polyoxyethylene hydrogenated castor oil, etc.) Then, a dispersant is added or, if desired, a base oil or fat is added to the dispersant using an emulsifier. Furthermore, these include a solubilizing agent (benzyl benzoate, etc.), alcohols (monovalent or polyvalent such as polyethylene glycol), glucose and other isotonic agents (for example, D-sorbitol) as necessary. , D-mannitol, sodium chloride, etc.), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.) ) Or an antioxidant such as ascorbic acid.
The chemical solution obtained in this way is filtered through a sterilization filter as necessary to make a sterile chemical solution, and it is usually aseptically filled into a suitable ampoule or the like, or filled into a vial as necessary. And it can be used for the medical field as a dried preparation for dissolution.

  Further, an additive component such as a stabilizer and a preservative suitable for a propellant may be selected from the components described in the previous section, and an absorption accelerator, an antistatic agent, and the like may be further added to form a liquid or dry fine powder. For topical spraying, the dosage form can be appropriately selected as a liquid spray or a fine powder or dry solid having a particle size suitable for pulmonary administration or nasal administration.

  Peptides are often cleared rapidly from the systemic circulation when administered, so that they exhibit their pharmacological activity only in a relatively short time. Therefore, in order to maintain a state effective for treatment, it is preferable to administer a biologically active substance in a relatively large amount and multiple times. Polyethylene glycol, copolymers of polyethylene glycol, and materials modified by covalently bonding water-soluble polymers such as polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, or polyproline are the corresponding unmodified It is known to show a longer half-life in blood after intravenous administration compared to these substances. Such modifications increase the solubility of the substance in aqueous solution, prevent aggregation, increase the physical and chemical stability of the substance, and may significantly reduce the substance's immunogenicity and reactivity. As a result, the desired in vivo bioactivity can be achieved by administering such polymeric substance adducts with fewer doses or in smaller doses compared to the unmodified substance.

  Since polyethylene glycol (PEG) has low toxicity, it is particularly useful to attach it. In addition, when PEG is bound, the immunogenicity and antigenicity of the heterologous compound may be effectively reduced. PEG reagents useful for reacting with amino groups of peptides include not only high-purity molecular weights of 2000 to 30000 but also activated PEG derivatives such as methoxy PEG amine, maleimide, carboxylic acid, etc. May be used for binding.

  The active substance of the prophylactic / therapeutic agent for inflammatory diseases of the present invention is a peptide having an anti-inflammatory action site in the amino acid sequence of human apolipoprotein A-II or a variant thereof, and has no or almost no side effects. It is a prophylactic / therapeutic agent for inflammatory diseases that exhibits extremely safe and reliable effects.

The present invention will be specifically described below with reference to examples and experimental examples.

Throughout the following experimental examples, examples and test examples, amino acids are represented by alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine. (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), and three letter display.
Experimental Example 1 (Synthesis of peptide)

  The peptides of SEQ ID NOs: 2 to 22 are prepared by using a peptide automatic synthesizer (type 433A: manufactured by Applied Biosystems) and binding amino acids one by one from the carboxyl terminal side to the amino acid derivative immobilized on the resin (solid phase). Synthesis).

The peptide represented by SEQ ID NO: 8 was synthesized as follows.
Fmoc-Ala-resin into which an amino acid (Ala) corresponding to the C-terminal residue of the peptide has been introduced is set in the reaction vessel of the peptide synthesizer, and as an activator according to the software attached to the synthesizer , 1- [bisdimethylaminomethylene] -1H-benzotriazolium-3-oxide-hexafluorophosphate (HBTu), 1-hydroxybenzotriazole (HOBt) and dimethylformamide (DMF) are added to the reaction vessel. And reacted. The obtained resin was gently stirred in piperidine-containing N-methylpyrrolidone to remove the Fmoc group. Next, in order to connect the second amino acid (Pro) from the C-terminal side, Fmoc-Pro-OH is activated by adding the same reagent as above, and bound to the resin except the previous Fmoc group. Reacted with amino acids. The Fmoc-Pro-Ala-resin produced here is again removed with the Fmoc group, reacted with an Fmoc-Gln (Trt) -OH / PyBOP solution activated in the same manner, and the third amino acid from the C-terminal side to the third amino acid. Fmoc-Gln (Trt) -Pro-Ala-resin was synthesized.

Furthermore, each amino acid was added sequentially in the same manner, and the desired protected peptide resin (Fmoc-Ala-Gly-Thr (tBu) -Glu (OtBu) -Leu-Val-Asn (Trt) -Phe-Leu-Ser ( tBu) -Tyr (tBu) -Phe-Val-Glu (OtBu) -Leu-Gly-Thr (tBu) -Gln (Trt) -Pro-Ala-resin).

The following amino acids were used for peptide synthesis. As these amino acids, L-form or D-form was used, and modified amino acids such as acetylation and pyrolation were also used.
The inside of () represents the protecting group which protects the reactive group of a residue part. :
Fmoc-Ala-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-His (Trt ) -OH, Fmoc-Ile-OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Asn (Trt) -OH, Fmoc-Pro-OH, Fmoc-Gln ( Trt) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Val-OH, Fmoc-Trp-OH, Fmoc-Tyr (tBu) -OH

The meanings of the abbreviations used in the above description are as follows. :
Trt = trityl,
Pbf = N-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
Boc = t-butoxycarbonyl,
tBu = tert-butyl,
OtBu = tert-butoxy HBTU = 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium-hexafluorophosphate

First, the protected peptide resin synthesized as described above (Ala-Gly-Thr (tBu) -Glu (OtBu) -Leu-Val-Asn (Trt) -Phe-Leu-Ser (tBu) -Tyr (tBu) The N-terminal Fmoc group was deprotected by piperidine treatment of) -Phe-Val-Glu (OtBu) -Leu-Gly-Thr (tBu) -Gln (Trt) -Pro-Ala-resin).

Next, it was washed with N-methyl-2-pyrrolidinone, then with methylene chloride, and further dried by blowing nitrogen gas.
The resin was taken out, added with a cleave solution, and reacted to remove the side chain protecting group and cut out the peptide from the resin. After the crude peptide consisting of the peptide of SEQ ID NO: 8 was ether precipitated, the precipitate was filtered. I took it.

In addition, a crude peptide solution containing the peptides described in SEQ ID NOs: 2 to 22 other than SEQ ID NO: 8 was also prepared by performing the same operations as these. The D-form peptide used was a D-form amino acid.
Experimental Example 2 (Peptide purification)

The crude peptide solution obtained in Experimental Example 1 was developed and purified by reverse phase chromatography using an ODS column (YMC ODS, 30 × 250 mm) with a linear concentration gradient of acetonitrile containing 0.1% trifluoroacetic acid. Fractions containing the product were collected and lyophilized to obtain a trifluoroacetate peptide having the amino acid sequence described in SEQ ID NOs: 2-22.
Experimental Example 3 (Production of C-terminal amide peptide)

Synthesis of the peptide represented by the peptide of 48-67 (C-terminal amide peptide) (SEQ ID NO: 11) was performed as follows. Using the same peptide automatic synthesizer as in Example 1, Rink-Amide MBHA for C-terminal amide production was used as a starting resin alone, and each Fmoc-amino acid derivative was used as a raw material according to the sequence from the C terminal to sequentially extend the peptide chain. The N-terminal Fmoc group was deprotected by piperidine treatment on the synthesized protected peptide resin, and then the side chain protecting group was removed and cleaved from the resin using trifluoroacetic acid to obtain a crude peptide solution. . Purification was performed in the same manner as in Experimental Example 2 to obtain the trifluoroacetate peptide of SEQ ID NO: 11. A crude peptide solution containing the peptides described in SEQ ID NOs: 12, 13, 15, 20, and 22 other than SEQ ID NO: 11 was also prepared by performing the same operations as these.
Experimental Example 4 (Preparation of disulfide bond-type cyclized peptide)

The peptide represented by SEQ ID NO: 12 was synthesized as follows. When the C-terminal is an amide using the same peptide automatic synthesizer as in Example 1, Rink-Amide MBHA for producing a C-terminal amide is used as a starting resin alone, and each Fmoc-amino acid derivative is sequentially used as a raw material according to the sequence from the C terminal. Was extended. The protected peptide resin obtained by synthesis was subjected to deprotection of the side chain protecting group and cleaving from the resin using trifluoroacetic acid. The obtained crude peptide solution was made into a 50% acetic acid aqueous solution, and an iodine aqueous solution was mixed and reacted to obtain a disulfide bond-type cyclized peptide. After confirming the progress of cyclization with a high performance liquid chromatograph, purification was performed in the same manner as in Experimental Example 2 to obtain the trifluoroacetate peptide of SEQ ID NO: 12. A crude peptide solution containing the peptides described in SEQ ID NOs: 13 and 16 other than SEQ ID NO: 12 was also prepared by performing the same operations as these.
Experimental Example 5 (Preparation of disulfide bond dimer peptide)

The peptide represented by SEQ ID NO: 21 was synthesized as follows. When the C-terminal is an amide using the same peptide automatic synthesizer as in Example 1, Rink-Amide MBHA for producing a C-terminal amide is used as a starting resin alone, and each Fmoc-amino acid derivative is sequentially used as a raw material according to the sequence from the C terminal. Was extended. The protected peptide resin obtained by synthesis was subjected to deprotection of the side chain protecting group and cleaving from the resin using trifluoroacetic acid. The obtained thiol group-free crude peptide solution was made into a 50% aqueous acetic acid solution, and an aqueous iodine solution was mixed and reacted to obtain a disulfide-bonded dimer peptide. After confirming the progress of dimerization with a high performance liquid chromatograph, purification was conducted in the same manner as in Experimental Example 2 to obtain the trifluoroacetate peptide of SEQ ID NO: 21. A crude peptide solution containing the peptide described in SEQ ID NO: 22 was also prepared by performing the same operations as these.
Experimental Example 6 (Salt exchange)

For the peptides represented by SEQ ID NOs: 4 and 11, the trifluoroacetate salt was dissolved in an acetic acid aqueous solution and salt-exchanged with a Dowex ion exchange resin to obtain an acetate salt.
Experimental Example 7 (Peptide purity)

The peptides obtained in Experimental Examples 1 to 6 were dissolved in PBS and used as peptide solutions as ODS columns (YMC ODS, 4.6 × 150 mm), (Inertsil ODS-3, 4.6 × 250 mm) or (Inertsil PREP ODS). , 4.6 × 250 mm) by reverse phase chromatography with a linear concentration gradient of acetonitrile containing 0.1% trifluoroacetic acid, and the absorbance was measured at 220 nm to confirm the purity.
The results are shown below.
The peptide of SEQ ID NO: 2 is 96.8%,
The peptide of SEQ ID NO: 3 is 95.9%,
97.9% of the peptide of SEQ ID NO: 4,
98.7% of the peptide of SEQ ID NO: 5,
92.1% of the peptide of SEQ ID NO: 6,
The peptide of SEQ ID NO: 7 is 95.4%,
The peptide of SEQ ID NO: 8 is 94.2%,
The peptide of SEQ ID NO: 9 is 95.9%,
95.4% of the peptide of SEQ ID NO: 10,
99.6% of the peptide of SEQ ID NO: 11,
The peptide of SEQ ID NO: 12 is 96.3%,
97.6% of the peptide of SEQ ID NO: 13,
98.6% of the peptide of SEQ ID NO: 14,
The peptide of SEQ ID NO: 15 is 98.9%,
97.5% of the peptide of SEQ ID NO: 16,
The peptide of SEQ ID NO: 17 is 98.4%,
The peptide of SEQ ID NO: 18 is 98.4%,
95.0% of the peptide of SEQ ID NO: 19,
The peptide of SEQ ID NO: 20 is 99.3%,
The peptide of SEQ ID NO: 21 is 99.2%,
The peptide of SEQ ID NO: 22 is 98.4%
As described above, high-purity peptides were obtained in all cases.
The chromatogram which measured the purity about the peptide of sequence number 2-22 was shown to FIGS. 1-21.

However, SEQ ID NO: 1 was produced according to International Publication Number WO / 2008/133225.
Experimental Example 8 (In vitro activity measurement)

"Test using human monocyte-like cell line THP-1-derived macrophages: MCP-1 production"
The physiological activity of the peptides (SEQ ID NOs: 2, 3, 10, 11, 12, and 13) was determined by measuring the production of MCP-1 under lipopolysaccharide (LPS) stimulation, It compared and evaluated as MCP-1 production inhibitory activity. Similarly, SEQ ID NO: 1 was also performed.
That is, the human monocytic cell line THP-1 was seeded in a 96-well plate at 2 × 10 ⁴ / well (100 uL / well), and at that time, phorbol 12-myristate 13-acetate (PMA) was added at a final concentration. It was added at 100 nM and cultured at 37 ° C. under 5% CO ₂ for 3 days. The medium was newly replaced with a PMA-containing medium (100 uL / well) and further cultured for 24 hours. After washing the cells, the peptide obtained in Experimental Examples 1 to 6 or PBS of solvent control was added at 50 uL / well, and LPS (E. coli 0111: B4, manufactured by Sigma) was added at a final concentration of 0 as a stimulus. The mixture was added at 50 uL / well to 1 μg / mL, and cultured at 37 ° C. under 5% CO ₂ for 9 hours or 24 hours. Then, MCP-1 (Human CCL2 / MCP-1 Quantikine ELISA Kit; manufactured by R & D) in the culture supernatant was measured.
The results are shown in Tables 1 and 2. The “value” in the following table is an average value (%) of n = 2.

As shown in Table 1, MCP-1 production was suppressed up to 15% in SEQ ID NO: 13 and up to 22% in SEQ ID NO: 11, and other peptides also showed MCP-1 production suppression compared to SEQ ID NO: 1. As shown in Table 2, SEQ ID NO: 3 showed dose-dependent inhibition of MCP-1 production in a concentration range of 0.01 to 0.125 mg / mL.

Experimental Example 9 (In vitro activity measurement)
“Test using rat microglia cells: TNF-α production”
The physiological activity of the peptides (SEQ ID NOs: 5, 7, 8, 9 and 11) was determined by measuring the production amount of TNF-α under stimulation with lipopolysaccharide (LPS) and comparing it with the production amount of PBS which had no effect. The TNF-α production inhibitory activity was evaluated. Similarly, SEQ ID NO: 1 was also performed.
That is, rat newborn-derived microglia cells were purchased from Sumitomo Bakelite Co., Ltd., and cultured for about a week according to the culture method of the company's Nerve-Cell Culture System <microglia>, and then used in the test system. After culturing, the microglia cells were suspended in a cell culture medium for microglia (Sumitomo Bakelite), seeded on a 96-well cell culture plate (Corning) at 5 × 10 ³ cells / well, and cultured overnight. The culture solution was replaced with a microglia test solution (Sumitomo Bakelite Co., Ltd.), and 50 μL / well of the peptide obtained in Experimental Examples 1 to 6 or solvent control PBS was added, and the final concentration was 25 ng / mL. E. coli 0111: B4 (manufactured by Sigma) was added at 150 uL / well and cultured at 37 ° C. in a CO ₂ incubator for 4 hours. The supernatant was collected, and TNF-α (Rat TNF-alpha Quantikine ELISA Kit; manufactured by R & D) in the supernatant was measured.
The results are shown in Tables 3 and 4.

As shown in Table 3, MCP-1 production was suppressed up to 0% in SEQ ID NO: 8, 1% in SEQ ID NO: 9, 18% in SEQ ID NO: 7, and 19% in SEQ ID NO: 11, and other peptides were also sequenced. Compared to No. 1, TNF-α production was suppressed. Further, as shown in Table 4, SEQ ID NO: 11 showed dose-dependent suppression of TNF-α production in a concentration range of 0.025 to 0.25 mg / mL.

Increased production of TNF-α, an inflammatory cytokine, is suppressed by the peptide preparation of the present invention using an inflammation model mouse developed using lipopolysaccharide (E. coli 0111: B4, manufactured by Sigma). confirmed.
First, the peptide (SEQ ID NO: 8) obtained in Experiments 1 to 6 was dissolved in PBS to obtain a clear solution having a concentration of 0.25 μg / ml. This was filtered with a 0.22 μm filter (Millipore) to prepare a peptide preparation.
For the experiment, eighteen 8-week-old male Balb / c mice (made by Charles River Japan) were prepared. Six of them were given a single intravenous dose of this peptide preparation as a peptide at a dose of 5 μg / kg (peptide administration group). Ten minutes later, the lipopolysaccharide solution 10 mg / kg was administered once intraperitoneally to a total of 12 animals, 6 animals in the peptide administration group and 6 animals in the other group (control group). On the other hand, 6 mice to which neither the peptide solution nor the LPS solution was administered were used as a normal group.
Two hours after administration of the peptide solution, blood was collected from the abdominal aorta under carbon dioxide anesthesia to obtain EDTA-containing plasma. The tumor necrosis factor (TNF-α) value in the plasma was measured, and the mean ± standard error was calculated for each. In the statistical analysis on the TNF-α value, Student's t-test (Excel, Microsoft) was performed for the effect of the control group on the normal group and the peptide administration group on the control group. The results are shown in FIG.
As shown in FIG. 22, in the control group, an increase in the TNF-α value in plasma was remarkably observed as compared with the normal group. However, the increase in TNF-α value observed in the control group was significantly suppressed in the peptide preparation administration group. In the peptide administration group, there were no side effects that seemed to be due to peptide administration.

The carrageenin-induced footpad edema model developed using λ-Carrageenan was used to examine the edema inhibitory effect of plasma-derived human apolipoprotein AII (pAPO) and apolipoprotein AII fragment peptide (SEQ ID NO: 8).
16 male ICR mice of 5 weeks old (manufactured by Charles River Japan Co., Ltd.) were prepared, 12 of which had a λ-Carrageenan (manufactured by Wako Pure Chemical Industries) solution prepared at 1.0 w / v% with physiological saline. The footpad of the left hind limb was locally administered at a dose of 50 μL / foot to induce a footpad edema model. Of these 12 animals, 4 animals were given a single intravenous administration of pAPO physiological saline solution as pAPO at a rate of 4 mg / kg 30 minutes before administration of the λ-Carrageenan solution (pAPO group). The physiological saline solution of No. 8 was administered once intravenously as a peptide at a rate of 5 μmg / kg (peptide group). The other 4 animals were not administered anything except λ-Carrageenan and served as a control group. About the remaining 4 animals, nothing was administered and they were set as normal groups. For these 16 animals, the swelling of the tarsal joint of the left hind limb was measured with calipers before λ-Carrageenan administration and 1, 3, 5 and 7 hours after administration, and the mean ± standard error was calculated respectively. The results are shown in FIG.
As shown in FIG. 23, swelling of the left hind limb was significantly observed in the control group. In contrast, the pAPO group showed an inhibitory effect from the 5th hour, but the peptide group showed an obvious inhibitory effect from the 1st hour, and the effect lasted until the 7th hour. In the peptide of this SEQ ID NO: 8 containing the amino acid of SEQ ID NO: 5 or SEQ ID NO: 22, effective reduction of inflammatory disease symptoms was confirmed, and the effect was increased in plasma-derived human apolipoprotein AII, which had a higher dose than the fragment peptide. Compared to a surprising level.

“Test using mouse CD4-positive T cells: IFN-γ production”
The physiological activity of the peptides (SEQ ID NOs: 4, 11 and 14) was determined by measuring the production amount of IFN-γ under stimulation with concanavalin A, and comparing the production amount with PBS, which had no effect, as IFN-γ production inhibitory activity. evaluated. Similarly, SEQ ID NO: 1 was also performed.
That is, the spleen was extracted from Balb / c mice to obtain spleen cells. Spleen cells were treated with FITC-labeled anti-CD4 antibody (Becton Dickinson), and CD4-positive T cells were isolated using FITC beads (Milteny). After seeding in a 96-well plate at 1 × 10 ⁶ / well (100 uL / well), various concentrations of peptide or solvent control PBS are added at 50 uL / well to give concanavalin A as a final concentration of 1 μg / mL. The mixture was added at 50 uL / well, and cultured at 37 ° C. under 5% CO ₂ for 24 hours. Thereafter, IFN-γ in the culture supernatant was measured by a specific ELISA (Mouse IFN-gamma Quantikine ELISA Kit; manufactured by R & D).
The results are shown in Tables 5 and 6.

As shown in Table 5, suppression of IFN-γ production by SEQ ID NO: 4 was 8%, SEQ ID NO: 11 was 12%, and SEQ ID NO: 14 was up to 18%. As shown in Table 6, SEQ ID NO: 4 showed dose-dependent inhibition of IFN-γ production in the concentration range of 0.25 to 1 mg / mL.

“Testing with human CD4-positive T cells: cell proliferation”
The physiological activity of the peptides (SEQ ID NOs: 10, 11, 13, 15, 20, and 22) was measured by cell proliferation activity under stimulation with anti-CD3 antibody and anti-CD28 antibody, and compared with the production amount in PBS, which had no effect. And evaluated as cell growth inhibitory activity. Similarly, SEQ ID NO: 1 was also performed.
That is, CD4-positive T cells were isolated from human blood using a CD4 + T cell isolation kit (Milteny). Anti-human CD3 antibody-immobilized 96-well plate was seeded at 5 × 10 ⁴ / well (100 uL / well), various concentrations of peptide or solvent control PBS were added at 50 uL / well, and anti-human was further stimulated CD28 antibody was added at 50 uL / well to a final concentration of 5 μg / mL, and the cells were cultured at 37 ° C. under 5% CO ₂ for 96 hours. Thereafter, cell proliferation activity was measured in the culture supernatant by the WST-1 method (Premix WST-1 Cell Proliferation Assay System; manufactured by Takara Bio Inc.).
The results are shown in Tables 7 and 8.

As shown in Table 7, the cell growth inhibitory activity of SEQ ID NO: 11 is 9%, SEQ ID NO: 20 is 11%, SEQ ID NO: 15 is 12%, and SEQ ID NO: 10 is 14%. Compared with 1, it showed cell growth inhibitory activity. As shown in Table 8, SEQ ID NO: 13 exhibited dose-dependent cell growth inhibitory activity in the concentration range of 0.03125 to 0.125 mg / mL.

"Test using mouse CD4-positive T cells: IL-2 production"
The physiological activity of the peptides (SEQ ID NOs: 4, 5, 6, 11 and 14) was determined by measuring the amount of IL-2 produced under concanavalin A stimulation and comparing it with the amount of production in PBS, which had no effect. Production inhibition activity was evaluated. Similarly, SEQ ID NO: 1 was also performed.
That is, the spleen was extracted from Balb / c mice to obtain spleen cells. Spleen cells were treated with FITC-labeled anti-CD4 antibody (Becton Dickinson), and CD4-positive T cells were isolated using FITC beads (Milteny). After seeding in a 96-well plate at 1 × 10 ⁶ / well (100 uL / well), various concentrations of peptide or solvent control PBS are added at 50 uL / well to give concanavalin A as a final concentration of 1 μg / mL. The mixture was added at 50 uL / well, and cultured at 37 ° C. under 5% CO ₂ for 24 hours. Thereafter, IL-2 in the culture supernatant was measured by a specific ELISA (Mouse IL-2 Quantikine ELISA Kit; manufactured by R & D).
The results are shown in Tables 9 and 10.

As shown in Table 9, SEQ ID NOs: 4, 5, and 11 showed up to 18% suppression of IL-2 production, and other peptides also showed IL-2 production suppression compared to SEQ ID NO: 1. Moreover, as shown in Table 10, SEQ ID NO: 14 showed dose-dependent suppression of IL-2 production in a concentration range of 0.25 to 1 mg / mL.

"Test using human CD4-positive T cells: IL-2 production"
The physiological activity of the peptides (SEQ ID NOs: 5, 7, 10, 11, 14, 16, 17, 18, 19 and 21) was measured by the amount of IL-2 produced under stimulation with anti-CD3 antibody and anti-CD28 antibody, It was evaluated as IL-2 production inhibitory activity in comparison with the production amount in PBS that had no effect. Similarly, SEQ ID NO: 1 was also performed.
That is, CD4-positive T cells were isolated from human blood using a CD4 + T cell isolation kit (Milteny). Anti-human CD3 antibody-immobilized 96-well plate was seeded at 5 × 10 ⁴ / well (100 uL / well), various concentrations of peptide or solvent control PBS were added at 50 uL / well, and anti-human was further stimulated CD28 antibody was added at 50 uL / well to a final concentration of 5 μg / mL, and the cells were cultured at 37 ° C. under 5% CO ₂ for 96 hours. Thereafter, IL-2 in the culture supernatant was measured by a specific ELISA (Human IL-2 Quantikine ELISA Kit; manufactured by R & D).
The results are shown in Tables 11 and 12.

As shown in Table 11, IL-2 production was suppressed up to 0% in SEQ ID NO: 11, 4% in SEQ ID NO: 10, 8% in SEQ ID NO: 18 and 14% in SEQ ID NO: 17, and other peptides were also sequenced. Compared to No. 1, IL-2 production was suppressed. Moreover, as shown in Table 12, SEQ ID NO: 11 showed dose-dependent suppression of IL-2 production in a concentration range of 0.0625 to 0.25 mg / mL.

  By using a small amount of this peptide for patients with inflammatory diseases accompanied by inflammatory cytokine production, chemokine production or T cell proliferation, prevention / treatment without side effects is possible.

Results of reverse phase chromatography confirming the purity of the peptide (SEQ ID NO: 2) Results of reverse phase chromatography confirming the purity of the peptide (SEQ ID NO: 3) Results of reverse phase chromatography confirming the purity of the peptide (SEQ ID NO: 4) Results of reverse phase chromatography confirming the purity of the peptide (SEQ ID NO: 5) Results of reverse phase chromatography confirming the purity of the peptide (SEQ ID NO: 6) Results of reverse phase chromatography confirming the purity of the peptide (SEQ ID NO: 7) Results of reverse phase chromatography confirming the purity of the peptide (SEQ ID NO: 8) Results of reverse phase chromatography confirming the purity of the peptide (SEQ ID NO: 9) Results of reverse phase chromatography confirming the purity of the peptide (SEQ ID NO: 10) Results of reverse phase chromatography confirming the purity of the peptide (SEQ ID NO: 11) Results of reverse phase chromatography confirming the purity of the peptide (SEQ ID NO: 12) Results of reverse phase chromatography confirming the purity of the peptide (SEQ ID NO: 13) Results of reverse phase chromatography confirming the purity of the peptide (SEQ ID NO: 14) Results of reverse phase chromatography confirming the purity of the peptide (SEQ ID NO: 15) Results of reverse phase chromatography confirming the purity of the peptide (SEQ ID NO: 16) Results of reverse phase chromatography confirming the purity of the peptide (SEQ ID NO: 17) Results of reverse phase chromatography confirming the purity of the peptide (SEQ ID NO: 18) Results of reverse phase chromatography confirming the purity of the peptide (SEQ ID NO: 19) Results of reverse phase chromatography confirming the purity of the peptide (SEQ ID NO: 20) Results of reverse phase chromatography confirming the purity of the peptide (SEQ ID NO: 21) Results of reverse phase chromatography confirming the purity of the peptide (SEQ ID NO: 22) Results of TNF-α measurement in LPS-induced inflammation model mice Inhibitory effect of carrageenin induced by peptide in edema-induced model mice

Symbol ** in FIG. 22: p <0.01 (for normal group)
##: p <0.01 (vs. control group)
Symbols in FIG. 23: normal group, ●: control group, x: pAPO group, ◆: peptide group

Claims (12)

A prophylactic / therapeutic agent for inflammatory diseases comprising a peptide consisting of 8 to 30 consecutive amino acids or a variant thereof in the peptide of SEQ ID NO: 2. The peptide consisting of 8 to 30 consecutive amino acids or a variant thereof is one that suppresses production of MCP-1, TNF-α, IFN-γ or IL-2, or suppresses T cell proliferation. The preventive / therapeutic agent for inflammatory diseases described. The inflammatory disease prophylactic / therapeutic agent according to claim 1 or 2, wherein the peptide comprising 8 to 30 consecutive amino acids comprises the peptide of SEQ ID NO: 23. The inflammatory disease prophylactic / therapeutic agent according to claim 1 or 2, comprising 20 to 30 consecutive amino acids including the peptide of SEQ ID NO: 4 or 5 in the peptide of SEQ ID NO: 2. Peptide variants are generated by substitution of one or more amino acids, deletion of one or more amino acids, addition of one or more amino acids, or linkage of SS bonds. The prophylactic / therapeutic agent for inflammatory diseases according to any one of the above. The inflammatory disease prophylactic / therapeutic agent according to claims 1 to 4, wherein the modified peptide is produced by N-terminal acetylation or pyrolation or C-terminal amidation. The prophylactic / therapeutic agent for inflammatory diseases according to claim 5, wherein the substitution of one or more amino acids is asparagine or serine substituted with alanine. The prophylactic / therapeutic agent for inflammatory diseases according to claim 5, wherein the substitution of one or more amino acids is generated by substitution of L-form amino acids with D-form amino acids. The prophylactic / therapeutic agent for inflammatory diseases according to claim 5, wherein the addition of one or more amino acids is made with a polar amino acid. The inflammatory disease prophylactic / therapeutic agent according to claim 9, wherein the polar amino acid is added via a spacer amino acid. The inflammatory disease preventive or therapeutic agent according to claim 1, wherein the inflammatory disease is accompanied by an increase in inflammatory cytokines, chemokines or T cells during inflammation. Diseases with increased inflammatory cytokines, chemokines or T cells may be systemic inflammatory response syndrome (SIRS), systemic vasculitis, bacterial osteomyelitis, dermatitis, psoriasis, diabetes, hepatitis, rheumatism, infection or autoimmunity Neuropathy associated with disease, neuropathic pain, ischemia reperfusion injury, encephalitis, Guillain-Barre syndrome, Parkinson's disease, arteriosclerosis, metabolic syndrome (heart syndrome), heart failure, cardiomyopathy, myocardial infarction, cerebral infarction, clone The prophylactic / therapeutic agent for inflammatory diseases according to claim 11, which is a disease comprising a disease, a graft host disease or a transplant rejection.