JP2000143694A - Activating factor for platelet-derived leukocyte phagocystosis sthenic factor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a polypeptide capable of forming a complex compound with transferrin or its derivative and manifesting a leukocyte phagocystosis sthenia by effecting a thrombin to an apolipo-protein C3. SOLUTION: This activating factor for a platelet-derived leukocyte phagocystosis sthenic factor is a polypeptide having an amino acid sequence of formula I (hereinafter called as PMA-II) or its pharmaceutically permissible salt. The PMA-II is obtained by effecting a thrombin that is a kind of protease, to an apolipo-protein. The PMA-II forms a complex compound with transferrin or its derivative, shows the phagocytic capacity-enhancing action of leukocyte and is utilized as a prophylactic or therapeutic agent of an infectious diseases, an immune-activating agent, an anti-thrombosis agent, etc. Also, polypeptides having amino acid sequences of formulae II to IV having one or several amino acids substituted, added or deficient in the PMA-II have antagonistic activities to the PMA-II and inhibit the leukocyte phagocystosis sthenia.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a polypeptide which promotes phagocytosis by leukocytes, a polypeptide having an antagonistic activity against the polypeptide, and a pharmaceutical composition containing each of these as an active ingredient.

[0002]

2. Description of the Related Art Leukocytes have a function of defending and cleaning a living body by ingesting and processing foreign cells such as microorganisms and unnecessary self cells such as waste products that have entered the body. This action is called phagocytosis or phagocytosis, and those having this function are called phagocytic cells or phagocytic cells. Among leukocytes, those having a phagocytic effect are neutrophils, monocytes, eosinophils and the like. Monocytes and neutrophils flow in the blood, infiltrate local inflammation, and exert phagocytosis. When monocytes leave the bloodstream and settle in a specific organ or the like, they mature and become macrophages. Macrophages are also phagocytic cells. Eosinophils also have a weak but phagocytic effect on bacteria.

[0003] The phagocytosis of these leukocytes can be divided into the following three in terms of their functions.

[0004] 1. Non-specific phagocytosis on foreign particles that have entered the living body In the immune antibody reaction, in the initial stage of infection until antibodies are produced, non-specifically, bacteria, fungi,
It engulfs foreign substances such as protozoa and viruses.

[0005] 2. Specific phagocytosis in the immune response Phagocytic cells feed on and destroy specific infectious microorganisms covered with IgG antibodies produced in response to infection,
It also plays an important role in the immune response.

[0006] 3. Treatment of damaged cells and waste autologous cells Phagocytic cells feed on not only foreign particles that have entered the living body as described above, but also damaged cells, dead autologous components such as aged blood cells and thrombus, and cancer cells. Therefore, if phagocytosis can be activated, it becomes possible to prevent and treat infectious diseases caused by pathogenic microorganisms that have entered the living body, improve immunity, and quickly remove thrombus. Phagocytic cells, on the other hand, cause inflammation during phagocytosis because they release cell-killing substances. For this reason,
From the viewpoint of prevention and treatment of excessive inflammation, it is desired to suppress phagocytosis. Thus, for phagocytosis by leukocytes, contradictory effects of activation / inactivation are required.

[0007] Among them, activation of phagocytosis by leukocytes mainly has the following two factors.

(1) Factors that activate in the presence of complement (2) Factors that activate via Fcγ receptor So far, in the case of (2), the molecular weight is 300,000 or 15
000 high molecular weight activators (MAPP: Macromolecular
Activators of Phagocytosis from Platelets), and it has been revealed that transferrin dimers or tetramers each occupy the main structure (Sakamot
o H., et al., Biochem. Biophys. Res. Common 230; 2
70-274, 1997).

[0009]

An object of the present invention is to provide a pharmaceutical composition capable of activating or inactivating the phagocytic activity of leukocytes.

[0010]

Means for Solving the Problems Recently, the present inventors have proposed that apolipoprotein C3 contained in platelets (the amino acid sequence and the like can be found in AshokV.
Hospattankar et al, FEBS Letters, vol.197, 1986,
(see p67-73) activates transferrin and enhances the phagocytosis of leukocytes (FIG. 1). However, apolipoprotein C3 alone did not enhance the phagocytosis of leukocytes.

Therefore, as a result of further intensive studies to solve the problems, the polypeptide obtained by allowing thrombin to act on apolipoprotein C3 forms a complex with transferrin or a derivative thereof, thereby enhancing the phagocytic activity of leukocytes. Was found to have. In addition, a polypeptide having antagonist activity of the polypeptide was also found, and the present invention was completed. That is, an object of the present invention is to provide the following polypeptides and pharmaceutical compositions containing them.

1. A polypeptide having the amino acid sequence of SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof.

2. A complex of the polypeptide or the pharmaceutically acceptable salt thereof according to the above 1 and transferrin or a derivative thereof.

3. A pharmaceutical composition having a leukocyte phagocytic activity-enhancing activity, comprising the polypeptide according to 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

4. A prophylactic or therapeutic agent for infectious diseases, comprising the polypeptide according to 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

5. An immunostimulator comprising the polypeptide of 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

6. An antithrombotic agent comprising the polypeptide according to 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[7] A polypeptide having an amino acid sequence in which one or several amino acids are substituted, added or deleted in the polypeptide according to the above 1, and having an antagonistic activity to the polypeptide according to the above 1, or a pharmaceutical thereof. Acceptable salts.

8. Among the polypeptides described in the above 7,
A polypeptide having the amino acid sequence of any one of SEQ ID NOs: 2, 3, and 4, or a pharmaceutically acceptable salt thereof.

9. A pharmaceutical composition comprising the polypeptide according to 7 or 8 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, which suppresses the effect of enhancing leukocyte phagocytosis.

10. An anti-inflammatory agent comprising the polypeptide of the above 7 or 8 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION The polypeptide in the present invention widely includes oligopeptides to proteins as long as they have the following activities.

1. Peptide having leukocyte phagocytosis-enhancing activity The polypeptide of the present invention includes a polypeptide having the amino acid sequence of SEQ ID NO: 1 (hereinafter, referred to as “PMA-II”). PMA-II forms a complex with transferrin or a derivative thereof (hereinafter, simply referred to as “complex”), and exhibits a phagocytic activity-enhancing activity of leukocytes.

[0024] PMA-II is, for example, apolipoprotein C3,
It can be obtained by allowing thrombin, a kind of protease, to act. Apolipoprotein C3 used
May be a commercially available product.For example, when platelets are subjected to ultracentrifugation, a high-density lipoprotein fraction can be obtained, and plasma high-density lipoprotein can be obtained by delipidation using a conventional method. Can be. When PMA-II is obtained by allowing thrombin to act on apolipoprotein C3, the two components may be simply mixed in a solvent. The solvent used in the reaction is not particularly limited as long as the generated PMA-II does not lose its activity. For example, ion-exchanged water, water such as distilled water, phosphate buffered saline (PBS), CPD solution (aqueous solution of citric acid, sodium citrate, glucose and sodium dihydrogen phosphate), Tris-HCl buffer, sodium acetate Aqueous solutions, buffers such as aqueous sodium citrate,
Organic solvents and the like may be mentioned, and a mixed solvent thereof may be used. Preferably, a buffer such as a PBS solution or a CPD solution having a pH of 6 to 8 is used. The reaction temperature is not particularly limited, but is usually 10 to 45 ° C, preferably 30 to 40 ° C. Although the reaction time is not particularly limited, it is generally 5 minutes to 2 hours, preferably 10 minutes to 1 hour. Purification of the solution after the reaction gives PMA-II. The purification method is not particularly limited as long as it is a method usually used in the art. It can be obtained by appropriately combining known polypeptide isolation and purification methods such as a polyethylene glycol fractionation method. For example, when subjected to anion exchange chromatography, it can be obtained as a non-adsorbed fraction. In addition, PMA-II can be obtained by conventional methods such as peptide synthesis, recombinant DNA technology, and point mutation as described below, in addition to the above methods. The obtained PMA-II may be purified by a method commonly used in the art.

The polypeptide of the present invention also includes a PMA-II derivative in which one or several amino acids are substituted, added or deleted in PMA-II, and which has an activity to enhance the phagocytic activity of leukocytes. When a complex with transferrin or a derivative thereof is formed, the PMA-II derivative is substituted, added or deleted in one or several of the amino acid sequence of PMA-II so as not to lose the leukocyte phagocytic activity-enhancing activity. Is a peptide. The leukocyte phagocytosis-enhancing activity of the PMA-II derivative may be qualitatively the same as that of PMA-II, and the degree is not limited. PMA-II like this
In order to obtain a derivative, the PMA-II should have a three-dimensional structure, a physicochemical property (for example, polarity / non-polarity, acidity / base of the constituent amino acids). One or several amino acids may be substituted, added or deleted. The PMA-II derivative can be obtained by conventional methods such as peptide synthesis, recombinant DNA technology, and point mutation as described below. The obtained PMA-II derivative may be purified by a method usually used in the art.

The PMA-II derivative may have an amino acid sequence in which one or several amino acids are substituted in PMA-II. The number of amino acids that may be substituted is not particularly limited as long as the complex does not lose phagocytic activity-enhancing activity, and is usually 1 to 3, preferably 1 to 2, and more preferably 1.

[0027] The PMA-II derivative may have an amino acid sequence obtained by adding one or several amino acids to PMA-II. The number of amino acids that may be added is not particularly limited as long as the complex does not lose the above activity, and is usually 1 to
7, preferably 1 to 5, more preferably 1 to 3.
In the case of a PMA-II derivative, the position of amino acid addition in PMA-II is not particularly limited as long as the complex does not lose its activity, but is preferably at the N-terminus or in the middle of the sequence of PMA-II.

The PMA-II derivative may have an amino acid sequence in which one or several amino acids have been deleted from PMA-II. The number of amino acids that may be deleted is not particularly limited as long as the complex does not lose the above activity, and is usually 1 to
3, preferably 1-2, more preferably 1. PMA-
In the case of the II derivative, the position of the amino acid deletion in PMA-II is not particularly limited as long as the complex does not lose its activity,
The N-terminal or the middle of the sequence of PMA-II is preferable.

The amino acids substituted or added in the polypeptides of the present invention (including PMA-II derivatives and PMA-II antagonists described below) are 20 amino acids (Gly, Ala, Ser, Val, Thr, Asn, G
ln, Pro, Glu, Asp, Leu, Ile, His, Met, Cys, Phe, T
L-form of yr, Trp, Lys and Arg), as well as the D-form of the above amino acids and special amino acids such as 4-hydroxyproline, N-methyllysine, β-alanine, N-methylglycine, N-methylasparagine, etc. .

The PMA-II and the PMA-II derivative may be protected or modified in the peptide main chain, side chain, terminal, etc., to such an extent that the complex does not lose its activity. Further, a complex protein may be formed and / or phosphorylated, such as a glycoprotein to which a sugar chain (monosaccharide, disaccharide, oligosaccharide or polysaccharide) is bound, or a lipoprotein to which lipid is bound. Good.

In the polypeptide of the present invention (including a PMA-II derivative and a PMA-II antagonist described below), the side chain which may be modified includes, for example, imidazole group, hydroxyl group, amino group, indole group, thiol group Group, thioether group, carboxyl group, carboxamide group,
Functional groups such as a guanidine group are exemplified, and preferred are a hydroxyl group, an amino group, and a carboxyl group. The protecting group which the polypeptide of the present invention may have is not particularly limited as long as it is a protecting group usually used in the art, and examples thereof include a C _1-6 acyl group such as a formyl group and an acetyl group. Can be In the polypeptide of the present invention, the carboxyl group and / or the amino group at the peptide terminal may be protected. Examples of the N-terminal protecting group include various acyl groups. Among them, acetyl (Ac), Fmoc,
Bpoc, trityl (Trt), Alloc and Boc are preferred. Examples of the C-terminal protecting group include various ester groups and substituted amino groups. Among them, esters with a C _1-6 alkyl group, an allyl group, an adamantyl group, a benzyl group and a t-butyl group, and —NH ₃ are preferable. Further, the main chain may be modified such that the nitrogen of the peptide bond is N-alkylated. The alkyl is preferably a methyl or ethyl group.

Further, the polypeptide of the present invention (including a PMA-II derivative and a PMA-II antagonist described below) may be a pharmaceutically acceptable salt. The salt is not particularly limited as long as the activity of the peptide of the present invention is not lost, and may be a salt usually used in the art. For example, inorganic acids (eg, hydrochloric acid, phosphoric acid, sulfuric acid, etc.), organic acids (eg, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, methanesulfonic acid, paratoluenesulfonic acid, etc.) ), Inorganic bases (eg, hydroxides and carbonates of alkali metals or alkaline earth metals such as sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate and sodium hydrogen carbonate), and organic bases. Can be.

Complex with transferrin or transferrin derivative Transferrin is a kind of β-globulin, which binds two molecules of ferric iron ions absorbed in blood to produce iron necessary for cell growth and production of hemoglobin. Is an iron-carrying protein that supplies intracellularly through a transferrin receptor. Transferrin or a derivative thereof used in the present invention is PMA-
There is no particular limitation as long as it forms a complex with II or a derivative thereof. Usually transferrin having a molecular weight of 150,000 or 300,000, and these transferrins may form dimers or tetramers as a form. Transferrin obtained by separating and purifying from human blood by a method known per se (for example, gel filtration) or a commercially available product can be used.

The transferrin derivative in the present invention is a transferrin polymer obtained by crosslinking transferrin with a crosslinking agent. Transferrin derivatives are
There is no particular limitation as long as it forms a complex with the polypeptide of SEQ ID NO: 1, and it is usually a dimer or tetramer. The crosslinking agent to be used is not particularly limited as long as it is a crosslinking agent generally used in the art, and examples thereof include dialdehydes such as glutaraldehyde, glyoxal, and succinic dialdehyde. The ratio between transferrin and the cross-linking agent in the transferrin derivative is not particularly limited as long as the activity of the complex is not lost.

The transferrin or a derivative thereof is PM
Forms a complex with A-II or a derivative thereof. The ratio of transferrin or its derivative to polypeptide in the complex depends on the type of PMA-II or its derivative and transferrin or its derivative used, and the concentration of each.
For example, the ratio of polypeptide to transferrin dimer or tetramer 1 is at least 1, usually 1 or more polypeptides per dimer and 2 or more polypeptides per tetramer.

When only the polypeptide is administered, PMA-II or a derivative thereof forms a complex with transferrin in the blood and exhibits a leukocyte phagocytosis-enhancing activity. Therefore, the polypeptide may be administered alone, but may be administered after forming a complex with transferrin or a derivative thereof in advance. In order to obtain the complex of the present invention, the two components may be simply mixed in a solvent in which PMA-II or a derivative thereof and transferrin or a derivative thereof do not lose their activities. The solvent for forming the complex is not particularly limited as long as it does not hinder the formation of the complex and does not lose the activity of the produced complex. For example, ion-exchanged water, water such as distilled water, phosphate buffered saline (PBS),
CPD solution (citric acid, sodium citrate, glucose and aqueous solution of sodium dihydrogen phosphate), Tris-HCl buffer, sodium acetate aqueous solution, sodium citrate aqueous solution and other buffers, organic solvents, and the like. There may be. Preferably, a PBS solution of pH 6 to 8, C
Buffer solution such as PD solution. The reaction temperature is not particularly limited, but is usually 10 to 45 ° C, preferably 30 to 40 ° C. The reaction time is not particularly limited, but is usually 10 minutes to 3 minutes.
Time, preferably 20 minutes to 2 hours.

Use as a preventive or therapeutic agent for infectious diseases Since the complex of the present invention has an action of enhancing leukocyte phagocytosis, it is effective in preventing or treating infectious diseases caused by pathogenic microorganisms such as bacteria, viruses and fungi, and for alleviating symptoms. It is. For example, even when there is a mucus layer or a capsule for resisting phagocytosis on the cell surface, the complex of the present invention promotes phagocytosis.
Examples of bacteria include Gram-negative bacteria such as Salmonella, Escherichia coli, and Pseudomonas aeruginosa; Gram-positive bacteria such as staphylococcus, streptococcus, and pneumococcus; and viruses such as herpes virus and HIV.
Fungi include Candida, Aspergillus and the like. Infections caused by these pathogenic microorganisms include acute gastroenteritis, food poisoning, urinary tract infections, cystitis, pyeloneitis, pneumonia, sepsis, staphylococci, skin diseases, herpes virus infections, herpes zoster, ringworm, candidiasis And the like.

Use as immunostimulant The complex of the present invention can be used for enhancing immunity. Specific examples include treatment of immunity decline after chemotherapy or radiation therapy, combined use with an antitumor agent, immunity decline due to AIDS-related syndrome, opportunistic infection and the like.

Use as an antithrombotic agent The complex of the present invention activates phagocytosis by leukocytes, thereby promoting the removal of waste products from the body. for that reason,
It can be used as an antithrombotic agent for the purpose of removing thrombus from atherosclerotic lesions.

2. Peptide having leukocyte phagocytosis-enhancing activity-suppressing activity The polypeptide of the present invention has an amino acid sequence in which one or several amino acids have been substituted, added or deleted in PMA-II (SEQ ID NO: 1), and A polypeptide having an antagonist activity against PMA-II (hereinafter, referred to as “PMA-II antagonist”) is also included.

The PMA-II antagonist activity of the PMA-II antagonist is an action of suppressing the leukocyte phagocytosis-enhancing action, and the degree of the activity is not limited. The ability to form a complex with transferrin is not limited.

The PMA-II antagonist may have an amino acid sequence in which one or several amino acids are substituted in PMA-II. The number of amino acids which may be substituted is not particularly limited as long as the antagonist activity to PMA-II is not lost, but is usually 1 to 3, preferably 1 to 2.

The PMA-II antagonist may have an amino acid sequence in which one or several amino acids are added to PMA-II. The number of amino acids that may be added is not particularly limited as long as the antagonist activity is not lost, but is usually 1 to 72, preferably 1 to 30.
It is. The site to be added is not particularly limited as long as the activity is not lost, but is usually at the C-terminus. PMA-II like this
As the antagonist, a polypeptide having a part of the amino acid sequence of apolipoprotein C3 is preferable, and polypeptides of SEQ ID NOs: 2 and 4 are particularly preferable. PMA-II
The amino acids to be substituted or added may be not only the 20 amino acids usually used in polypeptides but also the special amino acids as described above.

The PMA-II antagonist may have one or several amino acids deleted in PMA-II. The number of amino acids that may be deleted is not particularly limited as long as the antagonist activity is not lost, but is usually 1 to 3, preferably 1 to 2, and more preferably 1.
The site to be deleted is not particularly limited as long as the activity is not lost, but is usually at the C-terminus. As such a PMA-II antagonist, the polypeptide of SEQ ID NO: 3 is preferable.

Further, the PMA-II antagonist may be modified or protected on the peptide main chain, side chain, terminal and the like to the extent that the antagonist activity against PMA-II is not lost.
Further, it may form a complex protein such as a glycoprotein to which a sugar chain (monosaccharide, disaccharide, oligosaccharide or polysaccharide) is bound, a lipoprotein to which lipids are bound, and / or are phosphorylated. You may. Examples of the side chain that may be modified include the functional groups described above. PMA-II
The protecting group that the antagonist may have is not particularly limited as long as it is a protecting group usually used in the art, and examples thereof include the above-mentioned protecting groups.

Further, the PMA-II antagonist may be a pharmaceutically acceptable salt. The salt may be a salt commonly used in the art, as long as PMA-II antagonist activity is not lost. For example, the aforementioned acids and bases can be used.

The PMA-II antagonist can be obtained by a conventional method such as peptide synthesis, recombinant DNA technology, point mutation and the like. The obtained PMA-II antagonist may be purified by a method commonly used in the art.

-Utilization as an anti-inflammatory agent Phagocytic cells release their cell-killing substances during phagocytosis, thereby damaging their own tissues and causing inflammation.
Therefore, by suppressing phagocytosis, it is possible to prevent, treat and alleviate inflammation. Symptoms include acute inflammation due to sunburn or burns and chronic inflammation such as rheumatism.

Synthesis and Purification of Polypeptide When synthesizing the polypeptide of the present invention containing PMA-II and its derivatives and PMA-II antagonists, any of the commonly used solid phase synthesis method and liquid phase synthesis method may be used. And known methods may be combined. Alternatively, various commercially available peptide synthesizers may be used. The protecting group such as an amino group in peptide synthesis and the condensing agent in the condensation reaction are not particularly limited, and may be those usually used in the art. For example, Koichi Suzuki, "Protein Engineering-Fundamentals and Applications"
(1992, Maruzen Co., Ltd.), Bond Synthesis, et al., "Peptide Synthesis" (1976, John Wiley & So
ns, NY) and Stewart et al., Solid Phase
Peptide Synthesis "(1969, WH Freeman
and Co., San Francisco) can be used.

When the polypeptide of the present invention is prepared by recombinant DNA technology, host cells transformed with an expression vector containing a DNA encoding the polypeptide of the present invention are cultured in a medium according to a conventional method. It can be prepared by collecting the polypeptide of the present invention from the culture. Point mutation is another method for obtaining a substituted polypeptide. Examples of host cells include microorganisms (eg, bacteria (eg, Escherichia coli, Bacillus subtilis), yeast (eg, Saccharomyces)), animal cells (eg, COS cells,
Chinese hamster ovary (CHO) cells), insect cells (eg, Sf cells) and the like can be used. Known vectors such as Escherichia coli-derived plasmid, Bacillus subtilis-derived plasmid, yeast-derived plasmid, bacteriophage, and insect viruses such as baculovirus (nucleopolyhedrovirus) can be used as the vector.

When purifying the polypeptide of the present invention containing PMA-II and its derivative and PMA-II antagonist, for example, ion-exchange chromatography, ultrafiltration, gel filtration, affinity chromatography, adsorbent treatment, salt treatment, It can be obtained by appropriately combining known polypeptide isolation and purification methods such as precipitation, isoelectric point precipitation, and polyethylene glycol fractionation.

Dosage Form The PMA-II or a derivative thereof, a complex with transferrin or a derivative thereof and a PMA-II antagonist of the present invention may be a pharmacologically acceptable additive (eg, a carrier, an excipient, a diluent). , Stabilizing agents, etc.)
Pharmaceutical compositions in the form of powders, powders, granules, tablets, capsules, injections, sprays and the like, orally or parenterally (for example, intravenous, subcutaneous, intramuscular injections) according to a method known per se Suppositories or sublingual tablets).

An effective amount of the polypeptide or complex of the present invention is incorporated in the above preparation. The daily dose depends on the severity of the condition; age, sex, weight, and sensitivity of the subject;
Timing of administration, interval, route of administration, nature of pharmaceutical preparation, preparation,
Kind: It depends on the kind of the active ingredient, and is not particularly limited.
About 0.01 to 20 mg per body weight, preferably about 0.05
The dose is usually 10 to 10 mg, more preferably 0.1 to 5 mg, and is usually administered once or several times a day. When administered as a complex with transferrin or a derivative thereof, conversion may be performed again according to the molecular weight of transferrin or a derivative thereof.

As the above-mentioned pharmaceutically acceptable carrier, various organic or inorganic carrier materials commonly used as a preparation material can be used, such as excipients, lubricants, binders, disintegrants in solid preparations, and solvents in liquid preparations. , A diluent, a solubilizer, a suspending agent, an isotonic agent, a buffer, a soothing agent and the like. In addition, if necessary, pharmaceutical additives such as preservatives, antioxidants, coloring agents, sweeteners and the like can be used. Further, the stabilizer is not particularly limited as long as it is commonly used as a stabilizer for protein preparations.

For example, in the case of an injection, a diluent (physiological saline, distilled water for injection, sterile purified water, etc.), a suspending agent, a solubilizing agent, Stabilizing agents, isotonic agents, preservatives, dispersants, etc. are added to give aqueous or oily intravenous, subcutaneous, or intramuscular injections.
At that time, if necessary, a freeze-dried product can be obtained by a method known per se.

In order to prepare a preparation for oral administration, the polypeptide of the present invention is subjected to compression molding by adding, for example, an excipient, a disintegrant, a binder or a lubricant according to a method known per se, and then, if necessary. Oral preparations can be prepared by coating in a manner known per se for taste masking, enteric coating or long-lasting purposes.

In the case of an enteric preparation, an intermediate phase is preferably provided between the enteric phase and the drug-containing phase by a method known per se for the purpose of separating the two phases.

In order to prepare an external preparation, the polypeptide of the present invention can be made into a solid, semi-solid or liquid external preparation according to a method known per se. For example, as the solid, the polypeptide of the present invention is used as it is, or an excipient, a thickener and the like are added and mixed to obtain a powdery composition.
The liquid is almost the same as the injection, and is an oily or aqueous suspension. If semi-solid,
An aqueous or oily gel or an ointment is preferred.
In addition, any of these may include a pH regulator, a preservative, and the like. To make a spray, according to a method known per se,
The polypeptide of the present invention can be used as it is or as a buffer, stabilizer, sugar, deionized water, etc. (eg, 0.1 to 10 mg / ml)
And adjust the spray to be administered with a nebulizer.
The buffer preferably has a composition and molarity suitable for adjusting the solution to a pH in the range of 5-7. Nebulizer compositions provide a surface-induced aggregation (su) of proteins caused by atomizing the solution when forming an aerosol.
Conventional surfactants may be included to reduce or prevent rface induced aggregation).

[0059]

Industrial Applicability According to the present invention, a peptide (PMA-II) which enhances phagocytosis by leukocytes and a polypeptide having an antagonistic activity against the peptide have been found. That is, PM
A-II forms a complex with transferrin or a derivative thereof and can activate the phagocytic function by leukocytes. On the other hand, PMA-II antagonists can suppress the leukocyte phagocytosis-enhancing effect of PMA-II.

[0060]

EXAMPLES In the following, various experiments were conducted to explain the present invention in more detail. The present invention is not limited to these examples.

Example 1 Isolation and Purification of PMA-II Commercially available apolipoprotein C3 (Chemicon International)
Was added with thrombin (0.1 unit / ml) and reacted at 37 ° C. for 30 minutes. After the reaction, a MONO Q column (Pharmacia
Anion chromatography (eluent: 20 mM Tris-HCl buffer, pH 8.0) was used to obtain the target polypeptide as a non-adsorbed fraction. The non-adsorbed fraction was directly applied to a Protein sequencer (Applied Biosystem),
It was confirmed that the amino acid sequence was HATKTAK (SEQ ID NO: 1).

The apolipoprotein obtained by delipidating the plasma high-density lipoprotein using ether / alcohol, the frozen-thawed supernatant of preserved platelets, or the frozen-thawed supernatant of preserved platelets is further subjected to ultracentrifugation. Similar results were obtained when thrombin was allowed to act on the high-density lipoprotein fraction thus obtained. In addition, when apolipoprotein purified from stored platelets was passed through an affinity column for anti-apolipoC3 protein antibody, leukocyte phagocytosis-enhancing effect was observed in the adsorbed fraction.

Example 2 Formation of Complex by Transferrin or Its Derivative and Platelet-Derived PMA-II Synthesis and purification of transferrin derivative 0.2% glutaraldehyde was added to human-derived transferrin and reacted on ice for 2 hours. The obtained reaction solution is applied to a Superdex200 gel filtration column (elution buffer:
PBS, elution rate: 1 ml / min, fractionation volume: 1 ml), and the transferrin derivative was fractionated into a tetramer (TF4) and a dimer (TF2). Each fraction was subjected to native PAGE (7.5% polyacrylamide gel (manufactured by Bio-Rad), Tris-glycine buffer) to confirm that a tetramer and a dimer were formed, respectively.

2. Complex formation and its phagocytosis coefficient The obtained transferrin derivative (TF4 and TF2 are both 280n
(OD at m = 0.08), PMA-II prepared from platelets was added, and its phagocytic coefficient was determined (Table 1). In addition, a similar experiment was performed using transferrin with a molecular weight of 150,000 (pre-s-MAPP) or transferrin with a molecular weight of 300,000 (pre-l-MAPP) obtained by gel filtration of plasma while avoiding platelet stimulation. Was done.

The phagocytosis coefficient was determined by the method of Sakamoto H. et al. (J. Le
ukc. Biol. 50; 356-363, 1991), phagocytosis of immunoglobulin-sensitized sheep erythrocytes from neutrophils isolated from human peripheral blood on microplates was measured, and sheep erythrocytes were stimulated by PBS-stimulated neutrophils. The phagocytosis number was calculated as 100 (control). As shown in Table 1, in the case of PMA-II, transferrin or a derivative thereof alone, no phagocytic activity-enhancing effect was observed, and only when a complex was formed, a significant improvement in phagocytic activity was observed.

[0066]

[Table 1]

Example 3 Enhancement of phagocytic activity of human neutrophils by a complex with synthetic PMA-II The transferrin derivative obtained in Example 2 (TF4 and TF2 both OD at 280 nm = 0.08) was serially diluted. PMA-II was mixed with each other to form a complex. The phagocytic coefficient of human neutrophils at each concentration was determined, and the results in Table 2 were obtained.

[0068]

[Table 2]

Example 4 Enhancement of the phagocytic activity of human monocytes by the complex Human monocytes were isolated by the method of Sakamoto et al. (Br. J. Haematol., 57, 4).
7-60, 1984) and 10 mM of synthetic PMA-II and a transferrin derivative (TF2 or TF4 (OD at 280 nm = 0.0
8)) was obtained. The phagocytosis coefficient of the complex was calculated using the phagocytosis coefficient of monocytes stimulated by PBS as 100 (control) (Table 3). The complex with the transferrin derivative also promoted monocyte phagocytosis.

[0070]

[Table 3]

Example 5 Inhibition of Phagocytosis by PMA-II Antagonist Complex Production with Transferrin Derivative: Example 2
Transferrin derivatives (both TF4 and TF2)
(OD at 280 nm = 0.08), 100 pM of each polypeptide was added, and the mixture was reacted at 37 ° C. for 30 minutes to examine the complex-producing ability.

Inhibition of leukocyte phagocytosis-enhancing effect: The study was conducted by causing 1 mM of each polypeptide to act on platelet-derived MAPP. Platelet-derived MAPP was prepared by the method of Sakamoto et al. (Sakamoto H., et al., Biochem. Biophys. Res. Comm.
on 230; 270-274, 1997).

HATKTAKDALSSVQESQVAQQAR (SEQ ID NO: 2), H
ATKTA (SEQ ID NO: 3) and HATKTAKD (SEQ ID NO: 4) are both amino acid synthesizers (Multiple Peptide Synthesizer (S
It was chemically synthesized by Fmoc solid phase synthesis method using YRO II), MultiSyn Toc GmbH).

None of the polypeptides of SEQ ID NOs: 2 to 4 formed a complex with the transferrin derivative. In addition, all the polypeptides suppressed the function of MAPP having a leukocyte phagocytosis-enhancing effect (Table 4).

[0075]

[Table 4]

Formulation Example 1 (Injection) 1 mg of the polypeptide obtained in Example 1 was added to 1 ml of sterile purified water
And added human albumin as a stabilizer, pH
Was adjusted to 7.5. After filtering the bacteria, the mixture was filled in a vial and freeze-dried to prepare an injection.

[0077]

[Sequence List] SEQUENCE LISTING <110> Sakai Chemical Industry Co., Ltd. <120> Activation Factors of Phagocyctosis from Platelets <130> 2588JP <160> 4 <170> PatentIn Ver. 2.0 <210> 1 <211> 7 < 212> PRT <213> apolioprotein C3 <400> 1 His Ala Thr Lys Thr Ala Lys 1 5 <210> 2 <211> 23 <212> PRT <213> apolioprotein C3 <400> 2 His Ala Thr Lys Thr Ala Lys Asp Ala Leu Ser Ser Val Gln Glu Ser 1 5 10 15 Gln Val Ala Gln Gln Ala Arg 20 <210> 3 <211> 6 <212> PRT <213> apolipoprotein C3 <220> <400> 3 His Ala Thr Lys Thr Ala 1 5 <210> 4 <211> 8 <212> PRT <213> apolipoprotein C3 <220> <400> 4 His Ala Thr Lys Thr Ala Lys Asp 1 5

[Brief description of the drawings]

FIG. 1. Transferrin (usually dimer and tetramer)
It is taken up by platelets in the presence of calcium ions.
Thrombin is contained in PMA-I (apolipoprotein C) contained in platelets.
Acts on 3) to produce PMA-II. PMA-II forms a complex with transferrin and enhances the phagocytosis of leukocytes such as neutrophils and monocytes.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) A61P 37/06 A61K 31/00 637D A61K 38/00 C07K 14/535 C07K 14/535 14/775 14/775 14/79 14/79 C12P 21/06 // C12P 21/06 F term (reference) 4B064 AG01 CA21 CB01 CE11 DA03 DA08 4C084 AA02 AA07 BA01 BA17 BA18 BA41 CA59 DA01 MA13 MA17 MA23 MA35 MA37 MA41 MA43 MA63 MA66 ZA542 ZB022 ZB082 ZB092 ZB112 ZB322 ZC412 4H045 AA10 AA30 BA14 BA15 BA17 BA41 CA42 EA22 EA24 EA29 FA70 GA22 GA23

Claims (10)

[Claims] 1. A polypeptide having the amino acid sequence of SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof. 2. A complex of the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof and transferrin or a derivative thereof. 3. A pharmaceutical composition having a leukocyte phagocytosis-enhancing activity, comprising the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. 4. A preventive or therapeutic agent for an infectious disease comprising the polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. 5. An immunostimulator comprising the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. 6. An antithrombotic agent comprising the polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. 7. The polypeptide according to claim 1, wherein
A polypeptide having an amino acid sequence in which one or several amino acids have been substituted, added or deleted, and having an antagonistic activity to the polypeptide according to claim 1, or a pharmaceutically acceptable salt thereof. 8. A polypeptide having the amino acid sequence of any one of SEQ ID NOs: 2, 3, and 4, or a pharmaceutically acceptable salt thereof, among the polypeptides according to claim 7. 9. A pharmaceutical composition comprising the polypeptide of claim 7 or 8 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, which suppresses the effect of enhancing leukocyte phagocytosis. 10. An anti-inflammatory agent comprising the polypeptide according to claim 7 or 8 or a pharmaceutically acceptable salt thereof as an active ingredient.