JP4362780B2 - Peptides that inactivate anaphylatoxin C5a - Google Patents

Description

  The present invention relates to a novel peptide that inactivates C5a that acts as one of pathogenic substances including sepsis, DIC, multiple organ failure, allergic disease, ischemia reperfusion disorder, and the like, and a therapeutic agent.

  C5a is a glycoprotein consisting of 74 amino acids and having a molecular weight of about 11,000. It is generated by being decomposed into C5a and C5b by C4b2aC3b and C3bBbC3b which are C5 converters. This is generated by proteolytic cleavage of the N-terminus of C5, the fifth component of complement (Ntlsson et al., J. Immunol. 114: 815-822 (1975)). The biological properties of C5a extend through numerous cells and tissues involved in both acute and chronic inflammatory processes (Hugli, CRC Crit. Rev. Immunol., 1: 321-366 (1981)). Many of these properties are immunologically beneficial. C5a has been found to mediate host defense mechanisms in response to various pathological conditions. C5a is an inflammatory response, such as smooth microcontraction, increased vascular permeability, development of wheal and redness when injected into human skin, histamine release from mast cells, and oxidized cluster from polymorphonuclear leukocytes (PMNL) And is involved in a wide variety of specific biological functions normally associated with the induction of lysosomal enzyme release. C5a stimulates a measurable response from any circulating white blood cell, including basophils, eosinophils, monocytes and neutrophils (Hugli, supra; Bautsch et al., Immunobiol. 185: 41-52). (1992)). Furthermore, it has been discovered that a receptor (C5aR) for C5a exists in various cells such as vascular endothelial cells and nerve cells.

  Interaction of C5a with polymorphonuclear leukocytes and other target cells and tissues results in increased histamine release, vascular permeability, smooth muscle contraction, and inflammatory cells including neutrophils, eosinophils, and basophils (Hugli, TE, Springer, Semin. Immunopathol., 7: 193-219 (1981)). C5a also plays an important role in mediating the inflammatory effects of phagocytic mononuclear cells that accumulate at sites of chronic inflammation (Allison, A.C. et al., H.U. Agents and Actions, 8:27 (1978)). C5a can induce chemotaxis in monocytes and can cause monocytes to release lysosomal enzymes in a manner similar to the neutrophil response induced by these factors. C5a is known to have an immunoregulatory role, particularly at inflammatory sites, by enhancing the antibody response (Morgan, E.L. et al., J. Exp. Med., 155: 1412 (1982)).

  Therefore, control of C5a is expected as one of the treatment methods for vasculitis and multiple organ failure caused by a strong inflammatory reaction. Moreover, since C5a may act as an exacerbation factor of asthma and various allergic reactions, control of C5a is expected as one of the means of treatment. Furthermore, since C5a may be involved as an anaphylatoxin in various shock symptoms, the control of C5a is one of important treatment methods in that case. Therefore, the complementary peptide of the present invention having an action of inactivating C5a can be used as a therapeutic agent for various acute diseases including the above-mentioned pathological conditions. For this purpose, C5a receptor inhibitors have been developed as drugs that inhibit C5a, and antibodies against C5a have been developed as those that act directly on C5a to inhibit its action.

  For example, in the invention of JP-T-9-506258, the C5a analog is a C-terminal region of C5a (1-74): N′-Asn (64) -Ile (65) -Ser (66) -His (67 ) -Lys (68) -Asp (69) -Met (70) -Gln (71) -Leu (72) -Gly (73) -Arc (74) -C '(SEQ ID NO: 5) (C5a (1-74 ) Of amino acid sequences 64-) are disclosed. In Patent Document 2, the 37th to 53rd peptide (named PL37) from the amino terminus of C5a acts as an agonist of C5a, and when the peptide is loaded as a MAP peptide, it induces apoptosis in cells having C5a receptor. It has been found that it has an action of inducing the peptide, and a peptide consisting of the amino acid sequence of ASGAPAPGPAGPLRPMF (named mimetic "A") is reported as a complementary peptide to this part (PL37).

However, it is not possible to control C5a yet to suppress exacerbation factors of pathological conditions including sepsis, DIC, multiple organ failure, allergic disease, ischemia reperfusion injury, etc., and its appearance is desired.
JP-T 9-506258 JP 2004-269520 A

  The present invention relates to a novel peptide that directly binds to C5a and inhibits the activity of C5a, and pathologies involving C5a, sepsis, DIC (Disseminated Intrabascular Coagulation), multiple organ failure, allergic disease, Provide therapeutic agents for ischemia reperfusion injury and the like.

  In view of the above fact, the present invention uses, as a target peptide, an amino acid sequence of a peptide consisting of 17 amino acids from the 37th amino acid to the 53rd amino acid, which is considered to play a major role in the activity of C5a. A peptide having a high complementarity value is designed.

  The first feature of the present invention is a peptide that binds to anaphylatoxin C5a and inhibits the activity of C5a. The amino acid sequence is represented by 17 amino acids that are ASTAPARAGFPQMMQGL when expressed by the amino acid one-letter code (peptide 1 ).

  A second feature of the present invention is a peptide that binds to anaphylatoxin C5a and inhibits the activity of C5a. The amino acid sequence is represented by SEQ ID NO: 2 (peptide 2 ).

  A third feature of the present invention is a peptide that binds to anaphylatoxin C5a and inhibits the activity of C5a. The amino acid sequence is represented by SEQ ID NO: 3 (peptide 3), which is AYTMPGHAGIPRFTKML when expressed by the single letter code. ).

  A fourth feature of the present invention is a peptide that binds to anaphylatoxin C5a and inhibits the activity of C5a, and has an amino acid sequence of 17 amino acids that are ASTAPARAGLPRFTYTL when expressed by the single letter code. ).

  A fifth feature of the present invention is a peptide that binds to anaphylatoxin C5a and inhibits the activity of C5a. The amino acid sequence is represented by SEQ ID NO: 5 (peptide 5 ).

  A sixth feature of the present invention is a peptide that binds to anaphylatoxin C5a and inhibits the activity of C5a. The amino acid sequence is represented by SEQ ID NO: 6 consisting of 17 amino acids (APTMOGQTIYTQLMPDF) ).

  The seventh feature of the present invention is any one of claims 1 to 6, wherein one or two amino acids are deleted, substituted, modified or added in the amino acid sequence and have an action of inhibiting the activity of C5a. It is a peptide as described above. A peptide with higher activity and stability can be obtained by chemically modifying the peptide of the present invention or substituting an amino acid. Furthermore, although the peptide of the present invention can be used directly as a drug, a higher-order structure is analyzed using the peptide of the present invention as a prototype peptide, and a chemically synthesized drug having a similar structure is developed based on the knowledge obtained from the analysis. It can also be used.

  The eighth feature of the present invention is the prevention of diseases involving C5a containing at least one peptide according to any one of claims 1 to 7, which binds to anaphylatoxin C5a and inhibits the activity of C5a. It is a therapeutic agent. Examples of the pathological condition involving C5a include sepsis, DIC (Disseminated Intrabascular Coagulation), multiple organ failure, allergic disease, ischemia reperfusion disorder and the like. The present invention can provide a peptide that binds to anaphylatoxin C5a and inhibits the activity of C5a, and a preventive or therapeutic agent for a pathological condition involving C5a.

  The present invention provides a novel peptide that directly binds to C5a and inhibits the activity of C5a, and pathologies involving C5a, sepsis, DIC (Disseminated Intrabascular Coagulation), multiple organ failure, allergic It is possible to provide a preventive or therapeutic agent for a serious disease state such as a disease or ischemia / reperfusion injury.

  In order to save patients in the acute phase of the above-mentioned pathological conditions, it is necessary to administer a large amount of drug urgently. However, since prophylactic and therapeutic drugs containing the peptide of the present invention are rapidly decomposed in the body, administer a large amount. In addition, it is considered that if the administration is discontinued, it will rapidly decompose and disappear. Therefore, the drug accumulates and causes side effects and there are few concerns about the sequelae, and this drug can provide a useful means for saving a patient in an emergency. Furthermore, it has an effect that can be applied as a prototype for producing a chemically synthesized drug that simulates the higher-order structure of the peptide having a pharmacological effect and mimics the structure.

  The present invention will be specifically described below. The amino acids, three-letter code, and single-character code used in the present invention are shown in the following (). That is, there are 20 kinds of amino acids in nature, and these 20 kinds of amino acids can be divided into hydrophilic amino acids, hydrophobic amino acids and intermediate amino acids according to the structure of the alkyl group. Hydrophilic amino acids include glutamine (Gln; Q), glutamic acid (Glu; E), lysine (Lys; K), asparadine (Asn; N), aspartic acid (Asp; D), tylosin (Tyr; Y) and Histidine (His; H). Hydrophobic amino acids include valine (Val; V), leucine (Leu; L), isoleucine (Ile: I), phenylalanine (Phe; F), and methionine (Met; M). Intermediate amino acids include tryptophan (Trp; W), arginine (Arg; R), glycine (Gly; G), cysteine (Cys; C), serine (Ser; S), proline (Pro; P), alanine ( Ala; A), and threonine (Thr; T).

  It has been discovered that the 37th to 53rd peptides from the amino terminus of C5a act as agonists of C5a, and that when the peptide is loaded as a MAP peptide, it has the effect of inducing apoptosis in cells having the C5a receptor. Therefore, a complementary peptide for this portion was designed to obtain a peptide consisting of 17 amino acids.

  The peptide thus obtained is used as a preventive or therapeutic agent for pathologies involving C5a, sepsis, DIC (Disseminated Intrabascular Coagulation), multiple organ failure, allergic disease, ischemia reperfusion disorder, etc. When used as a pharmaceutically acceptable formulation comprising a therapeutically effective amount of one or more of the above peptides, one or more pharmaceutically acceptable carriers (additives) and / or dilutions It is prescribed with the drug. As described in detail below, the prophylactic and therapeutic agents of the present invention may be specifically formulated for administration in solid or liquid, including those adapted to: (1) oral administration , For example, liquid medicine (aqueous or non-aqueous solution or suspension), tablets, pills, powders, granules, paste for application to the tongue; (2) inhalants, dry powders and spray types, The particle size is changed by bronchial administration and intrapulmonary administration. (3) Parenteral administration, for example, as a sterile solution or suspension, for example, subcutaneous, intramuscular or intravenous injection, and other intracorporeal administration such as thoracic cavity, abdominal cavity, joint cavity, and cerebrospinal cavity; (4) local application E.g. as a cream, ointment or spray applied to the skin; or (5) in the vagina or rectum, e.g. as a vaginal suppository, cream or foam. However, in some embodiments, the compounds of the invention may simply be dissolved or suspended in sterile water.

  A “therapeutically effective amount” as used herein is an agent effective to produce any desired prophylactic, therapeutic effect at a reasonable benefit / risk ratio applicable to any medical treatment or It means the amount of the composition.

  “Pharmaceutically acceptable” means, within the scope of good medical judgment, no problems or complications such as excessive toxicity, irritation, allergic reaction, etc., commensurate with a reasonable benefit / risk ratio. In addition, it is used to refer to compounds, materials, compositions, and / or dosage forms suitable for use in contact with human and animal tissues.

  “Pharmaceutically acceptable carrier” as used herein is involved in carrying or transporting an agonist of the invention from one organ or part of the body to another organ or part of the body. Means a pharmaceutically acceptable material, composition or excipient, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the dosage form and not injurious to the patient.

  Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, trehalose and sucrose; (2) such as corn starch and potato starch. (3) Cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) cocoa butter and suppository waxes (9) Oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) glycerin, sorbitol, mannitol And polyethylene glycol (12) Esters such as ethyl oleate and ethyl laurate; (13) Agar; (14) Buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) Alginic acid; (16) Pyrogen free (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in drug formulations. In some embodiments, the drug formulation is non-pyrogenic. That is, the patient's body temperature is not increased.

The term “pharmaceutically acceptable salts” in this regard refers to the relatively non-toxic inorganic or organic acid addition salts of the compounds of the present invention. These salts may be prepared in situ during the final isolation and purification of the compounds of the invention, or the purified compounds of the invention can be separated from a suitable organic or inorganic acid in its free base form. And may be prepared by isolating the salt thus formed. Typical salts include hydrobromide, hydrochloride, sulfate, hydrogen sulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, lauric acid Salt, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionic acid Salt, and lauryl sulfonate. (See, for example, Berge et al. (1977), J. Pharm. Sci. 66: 1-19.)
Pharmaceutically acceptable salts of the agents of the present invention include the conventional non-toxic salts or quaternary ammonium salts of the compounds, eg, from non-toxic organic or inorganic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid; and acetic acid, propionic acid, succinic acid, Glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid There are salts prepared from organic acids such as toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isothionic acid and the like.

In other cases, the agents of the present invention may contain one or more acidic functional groups and are thus capable of forming a pharmaceutically acceptable salt with a pharmaceutically acceptable base. In these examples, the term “pharmaceutically acceptable salt” refers to a relatively non-toxic inorganic or organic base addition salt of a compound of the invention. These salts may also be prepared in situ during the final isolation and purification of the agent, or the purified agent in its free acid form, with a pharmaceutically acceptable metal cation. It can also be prepared by reacting separately with a suitable base, such as a hydroxide salt, carbonate or bicarbonate, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Good. Typical alkali or alkaline earth salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, and aluminum salts. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. (See, eg, Berge et al. (1977), supra).
Wetting agents such as sodium lauryl sulfate and magnesium stearate, emulsifiers and lubricants, and colorants, release agents, coatings, sweeteners, flavors and fragrances, preservatives and antioxidants are also present in the composition. May be.

  Examples of pharmaceutically acceptable antioxidants include: (1) Water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogensulfate, sodium disulfite, sodium sulfite and the like; (2 ) Oil-soluble antioxidants such as ascorbyl palmitate, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol and the like; and (3) citric acid, ethylenediaminetetraacetic acid (EDTA) ), Metal chelating agents such as sorbitol, tartaric acid, phosphoric acid and the like.

  Dosage forms of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. The dosage form may be conveniently presented in unit dosage form and may be prepared by any method well known in the pharmaceutical art. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect. Generally, out of 100 percent, this amount is the active ingredient ranging from about 1% to about 99%, preferably from about 5% to about 70%, most preferably from about 10% to about 30%.

  The methods of preparing these dosage forms or compositions comprise the step of combining one or more agents of the present invention with a carrier and optionally with one or more accessory ingredients. In general, dosage forms are prepared by uniformly and intimately bringing into association one or more agents of the present invention with liquid carriers or finely divided solid carriers or both and, if necessary, shaping the product.

  Suitable dosage forms of the invention for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (with seasoned active ingredients, usually sucrose and gum arabic or tragacanth), powders, granules, or As a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or pastilles (gelatin and glycerin, or inert groups such as sucrose and gum arabic) And / or a gargle and the like, each containing a predetermined amount of the compound of the present invention as an active ingredient. The agent of the present invention may be administered as a bolus, electuary or paste.

  In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), the active ingredient is one or more pharmaceuticals such as sodium citrate or dicalcium phosphate (1) a filler or bulking agent such as starch, lactose, sucrose, glucose, mannitol, trehalose and / or silicic acid; (2) For example, carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and / or gum arabic; (3) humectants such as glycerol; (4) agar, calcium carbonate, potato or tapioca starch, alginic acid, Certain silicates and decays like sodium carbonate (5) dissolution retardants such as paraffin; (6) absorption enhancers such as quaternary ammonium compounds; (7) wetting agents such as cetyl alcohol and glycerol monostearate; (8) of kaolin and bentonite clays; (9) Lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (10) colorants. In the case of capsules, tablets and pills, the drug composition may comprise a buffer. Similar types of solid compositions can also be used as fillers in soft and hard-filled gelatin capsules with excipients such as lactose or lactose and high molecular weight polyethylene glycols and the like.

  A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be binders (eg, gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (eg, sodium glycolate starch or crosslinked sodium carboxymethylcellulose), surfactants Alternatively, it can be prepared using a dispersant. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

  Solid dosage forms such as tablets of the drug composition of the present invention, such as dragees, capsules, pills and granules, are optionally scored or like coatings such as enteric coatings well known in the drug dispensing arts. May be prepared using a unique coating and shell. They use, for example, hydroxypropyl methylcellulose, other polymer matrices, liposomes and / or microspheres in various ratios to provide the desired release profile, and slow or controlled release of the internal active ingredient. May be formulated to provide. They may be sterilized, for example, by filtration through a bacteria retaining filter, or by incorporating a sterilant in the form of a sterile solid composition that can be dissolved in a sterile injectable medium such as sterile water immediately before use. . These compositions may optionally contain opacifiers, in compositions that release one or more active ingredients only in certain parts of the gastrointestinal tract or preferentially there, optionally in a delayed manner. There may be. Examples of embedding compositions that can be used are polymeric substances and waxes. The active ingredient may be in microencapsulated form, if appropriate, with one or more of the above-described excipients.

  Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. Liquid dosage forms include, in addition to the active ingredient, inert diluents commonly used in the art, such as water and other solvents, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzoic acid Like fatty acid esters of benzyl, propylene glycol, 1,3-butadiene glycol, oils (especially cottonseed oil, peanut oil, corn oil, embryo oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and sorbitan Solubilizers and emulsifiers, and mixtures thereof.

  In addition to inert diluents, oral compositions may include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, flavoring and preserving agents.

  Suspensions may be suspended in addition to the active compound, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof. A turbidity agent may be included.

  Dosage forms of the drug composition of the present invention for rectal or vaginal administration may be presented as suppositories. This suppository comprises mixing one or more agents of the present invention with one or more suitable nonirritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, suppository waxes or salicylates. Which is solid at room temperature but liquid at body temperature, it will melt in the rectum or vaginal cavity and release the active compound.

  The dosage forms of the present invention suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray dosage forms containing carriers as known to be suitable in the art.

  Dosage forms for topical or transdermal administration of one or more agents of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active agent may be mixed under sterile conditions with a pharmaceutically acceptable base material and, if necessary, preservatives, buffers, or propellants.

  Ointments, pastes, creams and gels are used in addition to the active compounds according to the invention for animal or vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide. Or an excipient such as a mixture thereof.

  Powders and sprays may contain, in addition to the compounds of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. The spray may further comprise customary high pressure gases such as chlorofluorinated hydrocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

  Transdermal patches have the additional advantage of controlled delivery of the agents of the present invention to the body. Such dosage forms can be made by dissolving or dispensing the compound of the present invention in the proper medium. Absorption enhancers can also be used to increase the flux of the agent of the invention across the skin. The speed of such flux can be controlled by either providing a speed control membrane or by dispersing the compound in a polymer matrix or gel.

  A pharmaceutical composition of the present invention suitable for parenteral administration is one or more pharmaceutically acceptable sterile isotonic or non-aqueous solutions, dispersions, suspensions together with one or more compounds of the present invention. Or an emulsion, or a sterile powder that can be reconstituted in a sterile injectable solution or dispersion just prior to use, which makes the antioxidant, buffer, bacteriostat, preparations isotonic with the blood of the intended recipient. Solutes, or suspending or concentrating agents.

  Examples of suitable aqueous and non-aqueous carriers that can be used in the drug compositions of the present invention include water, ethanol, polyols (eg, glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, such as olive oil. Vegetable oils, as well as injectable organic esters such as ethyl oleate. The inherent fluidity can be maintained, for example, by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersants and by the use of surfactants.

  These compositions may contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the activity of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbate and the like. It may be desirable to include isotonic agents such as sugars, sodium chloride in the composition. In addition, prolonged absorption of the injectable drug form can be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

  The compounds of the present invention may be given as such when administered to humans and animals as a medicament, or together with a pharmaceutically acceptable carrier, for example 0.1-99.5% (more preferably , 0.5-90%) as an active ingredient.

  The results of examining the effect of the peptide of the present invention on calcium influx caused by C5a stimulation are shown below, but this example is not intended to limit the scope of the invention described in the claims. .

  Human peripheral blood was collected from a vein, and neutrophils were separated using specific gravity centrifugation or the like. A fluorescence indicator (Fula2) was incorporated into the separated neutrophils, and the measurement of calcium influx occurring when C5a was allowed to act on the neutrophils was detected with an Hagamatsu photonics Argus apparatus.

  As a result, when 700 pM of C5a is allowed to act, calcium influx caused by C5a stimulation is detected as a fluorescent signal (FIG. 1: upper left). When 700 nM (FIG. 1: upper right) or 70 nM (FIG. 1: second left) of SEQ ID NO: 1 (peptide 1) was added to 700 pM C5a, the activity of C5a was completely suppressed. Compared with the control group of C5a, peptide 1 of SEQ ID NO: 1 was 44% active at 700 nM and suppressed (FIG. 1: third row).

  Human peripheral blood was collected from a vein, and neutrophils were separated using specific gravity centrifugation or the like. A fluorescence indicator (Fula2) was incorporated into the separated neutrophils, and the measurement of calcium influx occurring when C5a was allowed to act on the neutrophils was detected with an Hagamatsu photonics Argus apparatus.

  As a result, when 700 pM of C5a is applied, calcium influx caused by C5a stimulation is detected as a fluorescent signal (FIG. 2: upper left). When 700 nM (FIG. 2: upper right), 70 nM (FIG. 2: second left), or 7 nM (FIG. 2: second right) is added to this 700 pM C5a, C5a The activity of was suppressed. The activity of C5a was 47% when peptide 2 was added at 700 nM, 50% at 70 nM, and 87% at 7 nM. As a result, peptide 2 had a strong inhibitory effect at 70 nM or more, and a certain degree of inhibition was observed even when 7 nM was added (FIG. 2: third stage).

  Human peripheral blood was collected from a vein, and neutrophils were separated using specific gravity centrifugation or the like. A fluorescence indicator (Fula2) was incorporated into the separated neutrophils, and the measurement of calcium influx occurring when C5a was allowed to act on the neutrophils was detected with an Hagamatsu photonics Argus apparatus.

  As a result, when 700 pM of C5a is allowed to act, calcium influx caused by C5a stimulation is detected as a fluorescent signal (FIG. 3: upper left). When 700 nM (FIG. 3: upper right) or 70 nM (FIG. 3: second left) of SEQ ID NO: 3 (peptide 3) was added to 700 pM of C5a, the activity of C5a was suppressed. The activity of C5a was 14% when peptide 3 was added at 700 nM, and 74% when 70 nM was added. As a result, peptide 3 was found to have a very strong inhibitory action when added at 700 nM or more (FIG. 3: third stage).

  Human peripheral blood was collected from a vein, and neutrophils were separated using specific gravity centrifugation or the like. A fluorescence indicator (Fula2) was incorporated into the separated neutrophils, and the measurement of calcium influx occurring when C5a was allowed to act on the neutrophils was detected with an Hagamatsu photonics Argus apparatus.

  As a result, when 700 pM of C5a is applied, calcium influx caused by C5a stimulation is detected as a fluorescent signal (FIG. 2: upper left). 700 nM (FIG. 4: upper right), 70 nM (FIG. 4: left second stage), 7 nM (FIG. 4: right second stage), 700 pM (FIG. 4) are added to 700 pM C5a. 4, the third step on the left) suppressed the activity of C5a. The activity of C5a was 21% when peptide 4 was added at 700 nM, 24% at 70 nM, and 30% at 7 nM. As a result, it was confirmed that peptide 4 had a very strong inhibitory action at a concentration of 7 nM or more (FIG. 4: fourth stage).

  Human peripheral blood was collected from a vein, and neutrophils were separated using specific gravity centrifugation or the like. A fluorescence indicator (Fula2) was incorporated into the separated neutrophils, and the measurement of calcium influx occurring when C5a was allowed to act on the neutrophils was detected with an Hagamatsu photonics Argus apparatus.

  As a result, when 700 pM of C5a is applied, calcium influx caused by C5a stimulation is detected as a fluorescent signal (FIG. 2: upper left). When 700 nM (FIG. 5: upper right) or 70 nM (FIG. 5: second left) of SEQ ID NO: 5 (peptide 5) was added to 700 pM C5a, the activity of C5a was suppressed. The activity of C5a was 12% when peptide 5 was added at 700 nM and 88% at 70 nM. As a result, it was found that peptide 5 had a strong inhibitory action at 700 nM (FIG. 5: third stage).

  Human peripheral blood was collected from a vein, and neutrophils were separated using specific gravity centrifugation or the like. A fluorescence indicator (Fula2) was incorporated into the separated neutrophils, and the measurement of calcium influx occurring when C5a was allowed to act on the neutrophils was detected with an Hagamatsu photonics Argus apparatus.

  As a result, when 700 pM of C5a is allowed to act, calcium influx caused by C5a stimulation is detected as a fluorescent signal (FIG. 6: upper left). When 700 nM (FIG. 6: upper right) or 70 nM (FIG. 6: second left) of SEQ ID NO: 6 (peptide 6) was added to 700 pM C5a, the activity of C5a was suppressed. The activity of C5a was 66% when peptide 6 was added at 700 nM, and 81% at 70 nM. As a result, it was confirmed that peptide 6 had an inhibitory action at 70 nM or more (FIG. 6: third stage).

  From the above results, it was revealed that Peptide 1 to Peptide 6 each suppress the activity of C5a.

It is drawing which shows the effect | action of sequence number 1 (peptide 1) with respect to calcium influx which arises by the stimulation of C5a. It is drawing which shows the effect | action of sequence number 2 (peptide 2) with respect to calcium influx which arises by the stimulation of C5a. It is drawing which shows the effect | action of sequence number 3 (peptide 3) with respect to calcium influx which arises by the stimulation of C5a. It is drawing which shows the effect | action of sequence number 4 (peptide 4) with respect to calcium influx which arises by irritation | stimulation of C5a. It is drawing which shows the effect | action of sequence number 5 (peptide 5) with respect to the calcium influx which arises by the stimulation of C5a. It is drawing which shows the effect | action of sequence number 6 (peptide 6) with respect to the calcium influx which arises by the stimulation of C5a.

Claims (2)

  A peptide of SEQ ID NO: 4 (peptide 4) consisting of 17 amino acids which binds to anaphylatoxin C5a and inhibits the activity of C5a and whose amino acid sequence is represented by the one-letter code of amino acid, which is ASTAPARAGLPRFTYTL. A prophylactic or therapeutic agent for sepsis, DIC, multiple organ failure, allergic disease, ischemia reperfusion disorder involving C5a containing the peptide according to claim 1 that binds to anaphylatoxin C5a and inhibits the activity of C5a.

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