CN101233149A - Complement C3a derived peptides and uses thereof - Google Patents

Abstract

Peptides corresponding partially to positions 55-64 of the sequence of the complement component peptide C3a are capable of preventing and treating mast cell- and basophil-mediated disorders by inhibiting IgE- or IgG-mediated triggering and/or by inhibiting the FcepsilonRI- and/or FcgammaR-induced secretory response, while obviating the anaphylatoxic response. These peptides are useful for prevention and/or treatment of allergic disorders where mucosal-type and/or serosal-type mast cells and/or basophils are involved such as asthma, allergic dermatosis, and gastrointestinal allergies.

Description

Complement C 3 deutero-peptide and uses thereof

Technical field

The present invention relates to peptide derived from the aminoacid sequence of complement C 3, and in prevention and treatment by the allergic disorder of mastocyte or the basophilic granulocyte mediation pulmonary allergy purposes in the asthma for example particularly.

Background technology

Mastocyte and basophilic granulocyte play an important role in inflammatory and immediate hypersensitivity.The gathering that is present in the I type Fc epsilon receptor (Fc ε RI) in the plasma membrane of mastocyte and basophilic granulocyte has started the coupling cascade that reaches a climax in the inflammatory mediator secretion of (comprising histamine, serotonin, proteolytic enzyme, leukotriene and several cytokine).In the past few years, the molecular mechanism of being assembled the signal transduction that starts by Fc ε RI is furtherd investigate.The β subunit of src family protein Tyrosylprotein kinase (PTK) Lyn and receptor complex interacts, and carries out phosphorylation and activation owing to Fc ε RI assembles.Lyn is to the activation that causes Syk PTK of raising based on activation motif (the ITAM)-phosphorylation receptor subunits of immunity receptor tyrosine; the activation of Syk PTK causes that again Phospholipase C-γ (PLC-γ) activates; phosphatidyl-lipositol-4, the hydrolysis of 5-bisphosphate (PIP2) and free kytoplasm [Ca2+] _iInstantaneous increase.This has induced the activation of the protein kinase C that reaches a climax at last again in the medium secretion.

The mastocyte ancestors show as simple spectrum system, produce two kinds of different phenotypes when migrating to different tissues; So-called serous coat type (reticular tissue type) mastocyte is present in serous cavity, skin and the respiratory tract; The mucosal pattern mastocyte mainly is present in the gastrointestinal mucosa surface.However, the dependency differentiation organized of mastocyte is a reversible; The inoblast deutero-factor changes over the serous coat type with the mucosal pattern mastocyte, and IL-3 helps the mucous membrane phenotype.Except tissue distribution, the endocorpuscular medium content of life-span and cell thereof is also different.All express Fc ε RI for two types on its cytolemma, the gathering of Fc ε RI excites secretory reaction.

Different with the triggering that the Fc ε RI of mastocyte mediates, the Toplink approach of mast cells activation only takes place in serous coat type mastocyte.Set up the experimental model of serous coat type mastocyte by rat peritoneum or human skin mastocyte.For example P material or complement activation product C 3a and C5a trigger Toplink and stimulate (Mousli etc., Immunopharmcol.27:1-11,1995) by being exposed to polyamines or cationic peptide.The latter's complement deutero-anaphylotoxin is the most effective Toplink activator of (serous coat type) mastocyte secretory reaction.By contrast, the mucosal pattern mastocyte, for example rat basophilic myelocytic leukemia clone (RBL-2H3) is not replied described cationic peptide.According to proof, C3a and some derivative thereof suppress the IgE mediation the RBL-2H3 cell take off particle, and C5a does not have effect (Erdei etc., Int.Immunol.7:1433-1439,1995 to this process; Erdei etc., Immunol.Lett.68:79-82,1999).

C3a is unsuitable for as allergy preparations, and this is because it has irritated toxicity to serous coat type mastocyte, and promptly it can induce the medium secretion of mastocyte.

Authorize applicant's of the present invention United States Patent (USP) 6,682,740 disclose partially or completely peptide and the analogue thereof corresponding to the 50-77 position of people's complement derived peptide C3a sequence, and described peptide can suppress the triggering of IgE mediation and/or the secretory reaction of Fc ε RI inductive mucosal pattern mastocyte.

For can be used for preventing or the small peptide that takes off the relevant allergic disorder of particle of treatment and basophilic granulocyte and serous coat type and mucosal pattern mast cell mediated and the needs that contain the composition of described small peptide are not met yet.

Summary of the invention

Find according to the present invention, derived from can effectively suppressing the secretory reaction of Fc ε RI inductive mucosal pattern and serous coat type mastocyte and basophilic granulocyte and alleviate syndrome, and there is not the irritated toxic action of C3a with some peptide and the analogue thereof of part corresponding to the 55-64 amino acids sequence of human complement component C3a.

The restraining effect of having found these peptides provides the inhibition to the proximal event (as the protein phosphorylation of Fc ε RI β subunit and Tyrosylprotein kinase Lyn) of Fc ε RI irritant reaction coupling cascade, and to after the inhibition of incident (for example instantaneous increase of free cytoplasmic calcium ionic).

Part is disclosed first corresponding to the C3a derived peptide of the aminoacid sequence of the 55-64 position of human complement component C3a and passive systemic anaphylaxis and the symptoms of asthma that analogue can alleviate animal model thereof.

New peptides disclosed by the invention is derived from known human complement component C3a, and this human complement component C3a is the 77-mer peptide (SEQ ID NO:1) with following sequence:

Ser-Val-Gln-Leu-Thr-Glu-Lys-Arg-Met-Asp-Lys-Val-Gly-Lys-Tyr-Pro-Lys-Glu-Leu-Arg-Lys-Cys-Cys-Glu-Asp-Gly-Met-Arg-Glu-Asn-Pro-Met-Arg-Phe-Ser-Cys-Gln-Arg-Arg-Thr-Arg-Phe-Ile-Ser-Leu-Gly-Glu-Ala-Cys-Lys-Lys-Val-Phe-Leu-Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg-Arg-Gln-His-Ala-Arg-Ala-Ser-His-Leu-Gly-Leu-Ala-Arg。

The present invention relates to derived from the peptide with following SEQ ID NO:2 sequence of part corresponding to the 55-64 amino acids sequence of human complement c 3 a peptide:

Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg, also relate to its analogue, chemical derivative and salt, they can suppress the secretory reaction of cell, and wherein said cell is mucosal pattern mastocyte, serous coat type mastocyte and/or basophilic granulocyte; And wherein said secretory reaction is by the triggering that is selected from (i) IgE or IgG mediation and/or (ii) Fc ε RI or Fc γ R accumulative stimulate institute to induce.

On the one hand, the invention provides the peptide of the 55-64 amino acids sequence (SEQID NO:2) of derived from human complement C 3, or its analogue, chemical derivative or pharmacy acceptable salt, wherein said peptide has the aminoacid sequence of following general formula:

X1-Asp-X2-Asn-Tyr-Ile-Thr-X3 (SEQ ID NO:3 to 10),

Wherein

X1 is selected from hydrogen, low-grade alkane acidyl, Cys, Ser, D-Ala and D-Ala-D-Ala;

X2 is selected from Ser-Ser and Val-Val; And

X3 is selected from Arg, Arg-NH2, Glu-Cys-Arg and Glu-Cys-Arg-NH2;

Condition is, when X1 is hydrogen or low-grade alkane acidyl and X3 when being Arg or Arg-NH2, X2 is Val-Val so.

New peptides of the present invention can suppress to be selected from the secretory reaction of the cell of mucosal pattern mastocyte, serous coat type mastocyte and basophilic granulocyte.Described secretory reaction is by the triggering that is selected from (i) IgE or IgG mediation and/or (ii) Fc ε RI or Fc γ R accumulative stimulate institute to induce.

In certain embodiments, the invention provides peptide with the aminoacid sequence that is selected from following sequence:

(a)D-Ala-D-Ala-Asp-Ser-Ser-Asn-Tyr-Ile-Thr-Arg-Z(SEQ ID NO：7)；

(b)Ser-Asp-Ser-Ser-Asn-Tyr-Ile-Thr-Arg-Z(SEQ ID NO：11)；

(c)Asp-Val-Val-Asn-Tyr-Ile-Thr-Arg-Z(SEQ ID NO：12)；

(d)Cys-Asp-Ser-Ser-Asn-Tyr-Ile-Thr-Arg-Z(SEQ ID NO：13)；

(e)Ser-Asp-Ser-Ser-Asn-Tyr-Ile-Thr-Glu-Cys-Arg-Z(SEQ ID NO：14)；

(f) (a) and (b), (c), (d) or analogue (e);

(g) (a) and (b), (c), (d), (e) or chemical derivative (f); And

(h) (a) and (b), (c), (d), (e), (f) or salt (g),

Wherein Z represents to hold carboxylic acid, acid amides or alcohol.

Term D-Ala is meant the D isomer conformation of L-Ala.

According to some embodiment, peptide of the present invention has about 8 to about 12 amino-acid residues.In specific embodiment, peptide of the present invention has about 8 to about 10 amino-acid residues.

According to some embodiment, described peptide has the listed aminoacid sequence of SEQ ID NO:7, and wherein said Z is the carboxyl terminal acid amides.According to some embodiment, described peptide has the listed aminoacid sequence of SEQ ID NO:11, and wherein said Z is the carboxyl terminal acid amides.

According to the embodiment of some example, described Z is an acid amides, and described peptide is selected from following sequence:

(a) D-Ala-D-Ala-Asp-Ser-Ser-Asn-Tyr-Ile-Thr-Arg-NH2 is called as C3a32 (SEQ ID NO:7) herein;

(b) Ser-Asp-Ser-Ser-Asn-Tyr-Ile-Thr-Arg-NH2 is called as C3a31 (SEQ ID NO:11) herein;

(c) Asp-Val-Val-Asn-Tyr-Ile-Thr-Arg-NH2 is called as C3a14 (SEQID NO:12) herein;

(d) Cys-Asp-Ser-Ser-Asn-Tyr-Ile-Thr-Arg-NH2 is called as C3a29 (SEQ ID NO:13) herein;

(e) Ser-Asp-Ser-Ser-Asn-Tyr-Ile-Thr-Glu-Cys-Arg-NH2 is called as C3a35 (SEQ ID NO:14) herein.

According on the other hand, the invention provides pharmaceutical composition, it contains at least a peptide of the with good grounds principle of the invention as the 55-64 amino acids sequence of the derived from human complement C 3 of promoting agent, or its analogue, chemical derivative or pharmacy acceptable salt, and pharmaceutically acceptable carrier.According to some embodiment, the peptide in the described pharmaceutical composition has the aminoacid sequence that is selected from SEQ ID NO:3 to SEQ IDNO:14.In certain embodiments, described pharmaceutical composition has the listed aminoacid sequence of SEQ IDNO:7, and wherein carboxyl terminal is optional is the carboxyl terminal acid amides.In other embodiments, the peptide in the described pharmaceutical composition has the listed aminoacid sequence of SEQ ID NO:11, and wherein carboxyl terminal is optional is the carboxyl terminal acid amides.

According on the other hand, the invention provides at least a peptide or its analogue, chemical derivative or pharmacy acceptable salt and pharmaceutically acceptable carrier and be used for preventing and/or treating the purposes of the medicament of allergic disorder in preparation, wherein said peptide, its analogue, chemical derivative or salt have the aminoacid sequence that is selected from SEQ ID NO:3 to SEQ ID NO:14.In certain embodiments, described peptide has SEQ ID NO:7 or the listed aminoacid sequence of SEQ ID NO:11, and wherein carboxyl terminal is optional is the carboxyl terminal acid amides.According to some embodiment, described allergic disorder is the disease of basophilic granulocyte and/or mucosal pattern and/or serous coat type mast cell mediated.According to the embodiment of an example, described allergic disorder is an asthma.

According to another aspect, the invention provides the method that is used to prevent and/or treat allergic disorder, described method is included as the pharmaceutical composition that needs its curee administering therapeutic significant quantity, described pharmaceutical composition contains peptide or its analogue, chemical derivative or the pharmacy acceptable salt that is selected from the aminoacid sequence of SEQ ID NO:3 to SEQ ID NO:14 as having of promoting agent, and pharmaceutically acceptable carrier, prevention or treatment allergic disorder thus.According to some embodiment, described peptide is selected from SEQ ID NO:3 to 14.According to some embodiment, described peptide has SEQ ID NO:7 or the listed aminoacid sequence of SEQ ID NO:11, and wherein carboxyl terminal is optional is the carboxyl terminal acid amides.

In certain embodiments, described allergic disorder produces because of IgE or IgG mediated hypersensitivity (I type or III type) and/or Fc ε RI or the secretory reaction of Fc γ R inductive.According to other embodiments, described allergic disorder is by the cell type mediation that is selected from mucosal pattern mastocyte, serous coat type mastocyte and basophilic granulocyte.Can use the example of the allergic disorder of medicine composite for curing of the present invention and/or prevention to include but not limited to: allergic rhinitis comprises seasonal rhinitis and sinusitis paranasal sinusitis; Pulmonary disorder, for example asthma; Allergic skin disease, for example urticaria, angioedema, eczema, atopic dermatitis and contact dermatitis; Allergic conjunctivitis; Gastrointestinal allergy, for example food or drug-induced gastrointestinal allergy; Spasm; Feel sick; Vomiting; Diarrhoea; The irritability enteropathy; The eye transformation reactions; Cheilitis; Vulvitis; Uveitis; And anaphylaxis.According to the embodiment of an example, described allergic disorder is an asthma.

According on the other hand, the present invention relates to be used to prevent and/or treat the method for the allergic disorder that mediates by the cell type that is selected from serous coat type mastocyte and basophilic granulocyte, described method is included as the pharmaceutical composition that needs its curee administering therapeutic significant quantity, described pharmaceutical composition contains the peptide that is selected from following sequence as promoting agent, its analogue, chemical derivative and pharmacy acceptable salt, and pharmaceutically acceptable carrier:

(a) X1-Cys-Asn-R1-X4 (SEQ ID NO:15 to 20);

(b) X2-Lys-Val-Phe-Leu-Asp-X3 (SEQ ID NO:21 to 23); And

(c)X5-Asp-Ser-Ser-Asn-Tyr-Ile-R7(SEQ ID NO：24)；

Wherein

X1 is selected from hydrogen, low-grade alkane acidyl, Cys, Asp-Cys and Arg-Arg-Cys;

X2 is selected from hydrogen, low-grade alkane acidyl and Lys;

X3 is selected from

(i)Ala-Ala-Asn-R1-Ile-Thr-R2-Leu-R3-R4；

(ii) Cys-Cys-Asn-R1-Ile-Thr-R2-Leu-R3; And

(iii)Cys-Cys-Asn-R1-Ile-Thr-R2-Leu-R3-R4-Gln-His-R5-R6；

X4 is selected from Ile-Thr-R2-Leu-R3; And Ile-Thr-Arg-R7;

X5 is selected from low-grade alkane acidyl and Leu;

R1 is selected from the die aromatischen Aminosaeuren residue;

R2 is selected from Glu and Lys;

R3 is selected from positively charged amino-acid residue;

R4 is selected from Arg and Glu;

R5 is selected from Ala and Arg;

R6 is selected from Arg and Lys;

R7 is selected from hydroxyl (OH), Arg, Arg-NH2 and Agm (agmatine).

In other embodiments, described peptide is selected from following sequence:

(a)Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg(SEQ ID NO：25)；

(b)Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Arg(SEQ ID NO：26)；

(c) Asp-Ser-Ser-Asn-Tyr-Ile-Arg is called as C3a11 (SEQ IDNO:27) herein;

(d) Lys-Val-Phe-Leu-Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg is called as C3a4 (SEQ ID NO:28) herein;

(e) Lys-Lys-Val-Phe-Leu-Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg-Arg-Gln-His-Ala-Arg is called as C3a5 (SEQ ID NO:29) herein;

(f) Lys-Val-Phe-Leu-Asp-Ala-Ala-Asn-Tyr-Ile-Thr-Glu-Leu-Arg-Arg is called as C3a6 (SEQ ID NO:30) herein;

(g) Arg-Arg-Cys-Cys-Asn-Tyr-Ile-Thr-Arg-Arg is called as C3a10 (SEQ ID NO:31) herein;

(h) (a) and (b), (c), (d), (e), (f) or analogue (g);

(i) (a) and (b), (c), (d), (e), (f), (g) or chemical derivative (h); And

(g) (a) and (b), (c), (d), (e), (f), (g), (h) or salt (i).

In certain embodiments, described peptide has the arbitrary aminoacid sequence of SEQ ID NO:25 to SEQ ID NO:31, and wherein carboxyl terminal is optional is the carboxyl terminal acid amides.In specific embodiments, described peptide is selected from following sequence:

(a) Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg-NH2 is called as C3a7 (SEQ ID NO:25) herein;

(b) Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Arg-NH2 is called as C3a9 (SEQID NO:26 herein.

In certain embodiments, use contains the medicine composite for curing of the peptide that is selected from SEQ ID NO:15 to 31 and/or the allergic disorder of prevention, because of the secretory reaction of IgE or IgG mediated hypersensitivity (I type or III type) and/or Fc ε RI or Fc γ R inductive serous coat type mastocyte and/or basophilic granulocyte produces.The example of the allergic disorder that can be treated includes but not limited to: gastrointestinal allergy, for example food or drug-induced gastrointestinal allergy; Spasm; Feel sick; Vomiting; Diarrhoea and vulvitis.Should know United States Patent (USP) 6,682,740 disclose the method that is used for the treatment of the allergic disorder that the IgE mediated hypersensitivity (I type) that wherein relates to the mucosal pattern mastocyte causes, described method is included as the curee who needs it and uses peptide or its analogue or the derivative with the arbitrary aminoacid sequence of SEQ ID NO:15 to 31.The present invention discloses the method that is used for the treatment of or prevents wherein to relate to the allergic disorder of serous coat type mastocyte and/or basophilic granulocyte first, and described method is included as the curee who needs it and uses peptide or its analogue or the derivative with the arbitrary aminoacid sequence of SEQ ID NO:15 to 31.

According to accompanying drawing, explanation, embodiment and claim hereinafter, these and other embodiments of the present invention will better be understood.

The accompanying drawing summary

Figure 1A-C has described the inhibition ability of the synthetic peptide of the sequence with similar C3a stretching, extension to the secretory reaction of the RBL-2H3 cell of IgE mediation.

Figure 1A has described the C3a7 of different concns or C3a9 to the influence from the release of RBL-2H3 cell of the granzyme of IgE mediation, Hex.

Figure 1B has described C3a31, the C3a32 of 250 μ g or C3a35 to the influence from the release of RBL-2H3 cell of the Hex of IgE mediation.

Fig. 1 C has described C3a7 and the C3a9 influence to the TNF-α cytokine secretion of RBL-2H3.

Fig. 2 A-B has described C3a7 and the C3a9 inhibition to the tyrosine phosphorylation of the β subunit of Lyn, Fc ε RI and PI-3K.

Fig. 2 A, the general phosphorylation pattern of RBL-2H3 cell.

Fig. 2 B, Lyn, the β chain of Fc ε RI and the phosphorylation of PI-3K.Use anti--Actin muscle (end row) that all cells lysate is carried out immunoblotting, with the proof load albumen of same amount.

Fig. 3 has described peptide C3a7 and C3a9 to the free kytoplasm Ca in the mastocyte ²⁺The inhibition of ionic antigen induction increase.

Fig. 4 A-E has described the interaction of the beta chain of C3a and high-affinity IgE acceptor.

Fig. 4 A is by the C3a on the mastocyte of the specific antibody detection bone marrow derived of Western trace use Fc ε RI β chain and the covalent complex (swimming lane 1) of Fc ε RI β chain.In control sample (swimming lane 2), there is not C3a to exist.

Fig. 4 B and 4C have shown surface plasma body resonant vibration (SPR) measuring result: the biotinylated peptide of the 1st extracellular loop of fixing expression rat (B) and people (C) Fc ε RI β chain on the SPR-sensing chip; and real-time follow-up they with the interaction of C3a (combine and dissociate), as analyte.

Fig. 4 D, the confocal microscope image of use Cy3-IgE and the fluorescently-labeled RBL-2H3 cell of Cy5-C3a9.Shown the synthetic of the equator section (up) of typical cells and three optical sections at cell top (low row).

Fig. 4 E, FITC-C3a9 (donor) and and RBL-2H3 cell bonded Cy3-IgE (acceptor) between the histogram of FRET (fluorescence resonance energy transfer) (FRET) efficient.

The opposite sequence peptide C3a55 that Fig. 5 has described C3a31 and contrast thereof is to the influence of the blood histamine level of the mouse that is exposed to passive systemic anaphylaxis.

Fig. 6 has described by the C3a31 of the pulmonary function measurement of mouse asthmatic model and the opposite sequence peptide that the is called as C3a55 protective capability relatively of its contrast.

The detailed description of the invention

The present invention relates to the synthetic peptide based on the C terminal sequence of human complement c 3 a, its similar thing, chemical derivative and pharmaceutically acceptable salt. Described peptide can be used for suppressing the secretory reaction that the hypersensitivity (I type and III type) of IgE or IgG mediation and/or Fc ε RI or Fc γ R are induced, and wherein said reaction is by mucous membrane type and serous coat type mast cell and basophil born of the same parents mediation.

Peptide of the present invention derived from the 55-64 amino acids sequence of part corresponding to the listed human complement c 3 a of following SEQ ID NO:2: Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg, and similar thing, chemical derivative and pharmaceutically acceptable salt. The C end of peptide of the present invention can be its free carboxy form, or preferably, it can be amidated to increase the stability of described peptide, for example increases described peptide to the resistance of enzyme cracking in the organism. The carboxyl end can also be modified in the mode that increases its dissolving property.

Peptide of the present invention

The invention provides the new type of peptides of the mast cell that can be used for suppressing secretory reaction that Fc ε RI or Fc γ R induce and/or IgE or IgG mediation and basophil born of the same parents' hypersensitivity (I type or III type). Described mast cell comprises mucous membrane type and serous coat type mast cell.

According on the one hand, the invention provides peptide or its similar thing, chemical derivative or the acceptable salt of pharmacy derived from the 55-64 amino acids sequence (SEQ ID NO:2) of the human complement c 3 a of the amino acid sequence of following general formula I:

X1-Asp-X2-Asn-Tyr-Ile-Thr-X3 (SEQ ID NO:3 to 10);

Wherein

X1 is selected from hydrogen, low-grade alkane acidyl, Cys, Ser, D-Ala and D-Ala-D-Ala;

X2 is selected from Ser-Ser and Val-Val; And

X3 is selected from Arg, Arg-NH2, Glu-Cys-Arg and Glu-Cys-Arg-NH2;

Condition is, when X1 is hydrogen or low-grade alkane acidyl and X3 when being Arg or Arg-NH2, X2 is Val-Val so.

In certain embodiments, the invention provides the amino acid sequence that is selected from following sequence:

(a)D-Ala-D-Ala-Asp-Ser-Ser-Asn-Tyr-Ile-Thr-Arg-Z(SEQ ID NO：7)；

(b)Ser-Asp-Ser-Ser-Asn-Tyr-Ile-Thr-Arg-Z(SEQ ID NO：11)；

(c)Asp-Val-Val-Asn-Tyr-Ile-Thr-Arg-Z(SEQ ID NO：12)；

(d)Cys-Asp-Ser-Ser-Asn-Tyr-Ile-Thr-Arg-Z(SEQ ID NO：13)；

(e)Ser-Asp-Ser-Ser-Asn-Tyr-Ile-Thr-Glu-Cys-Arg-Z(SEQ ID NO：14)；

(f) (a), (b), (c), (d) or similar thing (e);

(g) (a), (b), (c), (d), (e) or chemical derivative (f); And

(h) (a), (b), (c), (d), (e), (f) or salt (g),

Wherein Z represents terminal carboxylic acid, acid amides or alcohol.

Term D-Ala refers to the D isomers conformation of alanine.

In one embodiment, peptide of the present invention is the SEQ ID NO:7 sequence that is accredited as the C3a32 peptide among the application: the peptide that D-Ala-D-Ala-Asp-Ser-Ser-Asn-Tyr-Ile-Thr-Arg-NH2 is listed, it is the 10-mer peptide of the 53-62 sequence of derived from human complement C 3 peptide, and wherein the carboxyl end is carboxyl end acid amides.

In another embodiment, peptide of the present invention is the listed peptide of SEQ ID NO:11:Ser-Asp-Ser-Ser-Asn-Tyr-Ile-Thr-Arg-NH2 that is accredited as the C3a31 peptide among the application, it is the 9-mer peptide derived from the 54-62 sequence of sequence human complement c 3 a peptide, and wherein the carboxyl end is carboxyl end acid amides.

In other embodiments, peptide of the present invention comprises the peptide that is called as C3a35 among the 9-mer peptide that is called as C3a29 among the peptide that is called as C3a14 among the application, the application and the application, and wherein the carboxyl end is Carboxylamide, and the sequence of aforementioned three kinds of peptides is:

C3a14：Asp-Val-Val-Asn-Tyr-Ile-Thr-Arg-NH2(SEQ ID NO：12)；

C3a29：Cys-Asp-Ser-Ser-Asn-Tyr-Ile-Thr-Arg-NH2(SEQ ID NO：13)；

C3a35：Ser-Asp-Ser-Ser-Asn-Tyr-Ile-Thr-Glu-Cys-Arg-NH2(SEQ ID NO：14)。

The present invention includes the salt of peptide of the present invention, fragment, similar thing and chemical derivative. As used herein, term " salt " refers to carboxylic salts and the amino acid addition salt of peptide molecule. Carboxylic salts can generate by methods known in the art, comprises inorganic salts, such as aluminium, ammonium, calcium, copper, iron, ferrous iron, lithium, magnesium, manganese, inferior manganese, potassium, sodium, zinc etc. The salt that comprises following material derived from pharmaceutically acceptable organic salt without toxic bases: primary amine, secondary amine and tertiary amine; Replace amine, comprise naturally occurring replacement amine; Cyclammonium; And basic ion exchange resin, for example arginine, beet alkali, caffeine, choline, N, N '-dibenzyl ethylenediamine, diethylamine, 2-diethylaminoethanol, DMAE, ethylaminoethanol, ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucose amine, histidine, Kazakhstan amine, isopropylamine, lysine, cardiografin, morpholine, piperazine, piperidines, polyamines resin, procaine, purine, cocoa alkali, triethylamine, trimethylamine, three propylamine, tromethamine etc.).

Acid-addition salts comprises the salt of inorganic acid, and described inorganic acid is acetic acid, benzene sulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, second sulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, hydroxyl second sulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, glactaric acid, nitric acid, two hydroxyl naphthalene acid, pantothenic acid, phosphoric acid, butanedioic acid, sulfuric acid, tartaric acid, p-methyl benzenesulfonic acid for example.

Term as used herein " peptide " is intended to comprise the amino acid residue by interconnective natural, the non-natural of peptide bond or non-peptide bond and/or chemical modification. In whole specification and claim book, as known in the art, represent amino acid residue by one or three alphanumeric codes. Compound of the present invention comprises linear peptide and cyclic peptide and derivative and similar thing.

It is not the peptide of the other chemical part of a peptide molecule part that this paper employed " chemical derivative " refers to contain usually, and described chemical part is ester and the acid amides of free carboxy for example, free amino acyl group and alkyl derivative, ester and the ether of free hydroxyl. Target amino acid residue by making peptide is introduced described modification in the described peptide with can reacting with organic agent of deriving of selected side chain or the reaction of terminal residue.

Peptide analogues comprises 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor and/or the interpolation of using natural or alpha-non-natural amino acid residue. Peptide analogues comprises peptide simulation thing. Peptide simulation thing or " plan peptide " are simulating peptide biologically actives but essence not exclusively is the molecule of peptide. No matter be non-peptide wholly or in part, plan peptide according to the present invention provide approximate this plan peptide based on the space of the three-dimensional of group of the peptide chemical part of arranging arrange. Because this similar avtive spot structure is intended peptide biological system is had impact, this is similar to the biologically active of peptide.

The salt of described peptide, similar thing and chemical derivative be preferred for changing described peptide with such as the relevant pharmaceutical properties such as stability, solubility.

Pharmaceutical composition

The present invention also comprises pharmaceutical composition, and described pharmaceutical composition contains peptide of the present invention, its similar thing, chemical derivative or pharmaceutically-acceptable salts, and pharmaceutically acceptable carrier.

Do not wish to interrelate with any theory, the inferior unit of Fc ε RI-β can regulate or regulate and control the signal conduction of Fc ε RI and Fc γ R or activate (referring to such as Dombrowicz etc., Immunity 8:517-529,1998). Therefore, the present invention includes the allergic disease that the secretory reaction by mast cell and/or basophil born of the same parents causes, as known in the art, described secretory reaction is regulated by the mediation of FcR hypotype and by the inferior unit of Fc ε RI-β.

Except other consideration factors, the fact that novel active composition of the present invention is peptide, peptide analogues, peptide derivant or its salt requires described preparation should be suitable for sending the compound of these types. Under the normal conditions, peptide seldom is suitable for oral administration, and this is because it is responsive to hydrochloric acid in gastric juice or intestines enzyme, takes administration but disclose now composition palatable according to the present invention. Pharmaceutical composition of the present invention can pass through any suitable administration, for example local, nose is interior, subcutaneous, in the muscle, in the vein, in the artery, in the joint, damage outside interior or the stomach and intestine. Comprise within the scope of the invention by inhalation.

Peptide of the present invention as active component uses known pharmaceutically acceptable and compatible with described active component diluent or excipient dissolving, disperses or mixes. Suitable carrier or excipient are such as water, salt solution, phosphate buffered salt solution (PBS), glucose, glycerine, ethanol etc. and combining form thereof. Other suitable carriers are for well known to a person skilled in the art. In addition, if necessary, described composition can contain auxiliary agent in a small amount, for example wetting agent, emulsifying agent, pH buffer, stabilizing agent, adhesive (such as polyethylene pyrroles ketone, gelatin, hydroxypropyl methyl fiber element), lubricant, disintegrant (such as primojel, crosslinked polyethylene pyrroles ketone, crosslinked carboxymethyl cellulose sodium), surfactant, thickener, antioxidant etc.

Pharmaceutical composition of the present invention can prepare by means commonly known in the art, for example mixing, the dissolving by routine, granulates, mills, grinds powder, sugaring clothing, levigate, emulsification, encapsulate, embedding or freeze drying process.

The pharmaceutical composition of using according to the present invention can use on one or more physiology acceptable carrier or excipient (comprising auxiliary agent) to prepare by conventional method, and they can promote described active component to be processed into the preparation that can pharmaceutically use. Suitable preparation depends on selected method of administration.

For passing through inhalation, can adopt the pressurization bag that contains or do not contain suitable propellant or the aerosol spray form of sprayer to send according to pharmaceutical composition of the present invention, described propellant is dicholorodifluoromethane, trichlorine fluoromethane, dichlorotetra-fluoroethane or carbon dioxide for example. In the situation of pressurized aerosol, can determine dosage unit by the valve of sending measured quantity is provided. Can prepare capsule and the cartridge case (as being used for sucking the gelatin of device or insufflator) of the mixture of powders that contains described peptide and suitable powder matrix (such as lactose or starch).

The pharmaceutical composition that can orally use comprises the soft encapsulation capsule that slippage (push-fit) formula capsule and gelatin and plasticizer (such as glycerine or sorb sugar alcohol) that gelatin is made are made. The formula capsule of slippaging can contain with such as the filler of lactose, the active component that mixes mutually such as the adhesive of starch, such as the lubricant of talcum or stearic acid magnesium and optional stabilizing agent. In the flexible glue capsule, reactive compound solubilized or be suspended in suitable liquid such as fat oil, liquid paraffin or liquid polyethylene glycol. In addition, can add stabilizing agent. The preparation of the oral administration that is useful on can be the dosage that is suitable for selected method of administration. For oral administration, described composition can adopt with the tablet of conventional method preparation or the form of lozenge.

Lozenge nuclear uses suitable dressing to provide. Purpose can be used concentrated sugared solution for this reason, and described sugared solution can be chosen wantonly and contain Arabic gum, talcum, polyvinylpyrrolidone, poly-carboxylic ethene glue, polyethylene glycol, titanium dioxide, lacquer solution and suitable organic solvent or solvent thing. Can add dyestuff or pigment to tablet or lozenge dressing to be used for identification or to characterize the various combination of active compound doses.

For injection, can prepare compound of the present invention with the aqueous solution, preferably use buffer solution compatible on the physiology such as Han Keshi solution, woods grignard solution or normal saline buffer solution. For mucosal, in preparation, use the bleeding agent that is suitable for penetration barriers. Bleeding agent such as polyethylene glycol are known in this field.

The pharmaceutical composition of parenteral comprises the aqueous solution of the active component of aqueous solution form. In addition, can be suitable oil-containing injectable suspensions with the suspension preparation of reactive compound. Suitable natural or synthetic carrier is (Pillai etc., Curr.Opin.Chem.Biol.5,447,2001) known in this field. Optional ground, described suspension also can contain suitable stabilizing agent or the reagent of the solubility that increases active component, to allow the highly concentrated solution of preparation. But selection of land, described active component can be powder type, uses before use suitable carrier such as aseptic apirogen water heavy molten.

Pharmaceutical composition of the present invention can also use for example conventional suppository matrix (for example cocoa fat or other glyceride) with the preparation of rectal compositions form, for example suppository or enema,retention.

Be applicable to pharmaceutical composition of the present invention and comprise the composition that wherein contains active component with the amount of effective acquisition expection purpose. More particularly, " treatment effectively amount " meaning is effectively to prevent, postpone, alleviate or to alleviate the amount of compound of symptom of curee's allergic disease. Treating effective amount fixes in those skilled in the art's the limit of power really.

The toxicity of peptide as herein described and similar thing, derivative or salt and therapeutic efficiency can determine by the standard pharmaceutical procedures of cultivating in thing or the animal used as test at cell, for example the IC50 (concentration of 50% inhibition is provided) by measuring tested peptide. The dosage range that can be used for being identified for the people from the data of these cell culture assays and zooscopy acquisition. Described dosage can be according to the method for administration of applied formulation and use and is different. Each doctor can select definite preparation, method of administration and dosage (such as Fingl etc., 1975, the 1st chapter is the 1st page in " The Pharmacological Basis of Therapeutics ") according to patient's illness.

According to the seriousness of illness to be treated, administration can also be the single-dose of slow releasing composition, continues several days the course for the treatment of to several weeks or until obtains to cure or obtain weakening of morbid state. Certainly, the amount of composition to be administered can be depending on curee's immune state and health status, the seriousness of disease or illness, medication, or other relevant factors.

Preparation and medication be intended to the explanation and unrestricted. Should be understood that and use instruction content provided herein, can design easily other suitable preparation and administering modes.

According to certain embodiments of the present invention, the peptide that C3a derives or the treatment of similar thing effectively amount are at the dosage of about 0.02mg/kg to the scope of about 10mg/kg. Preferably, the dosage of described peptide of the present invention, derivative or similar thing is in the scope of about 0.05mg/kg to about 2mg/kg, and more preferably, the dosage of described peptide, derivative or similar thing is in the scope of about 0.1mg/kg to about 1mg/kg. Should understand described dosage and can be ascending-dose, so that can at first give low dosage, then give higher dosage, until obtain suitable reaction. In addition, can be in the treatment process with multiple dosing with the dosed administration of described composition in the curee, wherein when each administration, give the part of described dosage.

In certain embodiments, peptide of the present invention and derivative thereof and similar thing as modified delivery of peptides to cell. In one embodiment, peptide of the present invention is connected in cell-penetrating peptides (CPP). In a preferred embodiment, CPP is the amino acid sequence that contains fruit bat rqikiwfqnrrmkwkk (ANTP) structure territory or its fragment.

Therapeutical uses

Peptide of the present invention and similar thing, chemical derivative and salt can be used for pharmaceutical composition or the medicament for the preparation of preventative or the mammiferous allergic disease of therapeutic treatment.

The present invention relates to for preventing and/or treat the method for not induced irritated toxic reaction by the allergic disease of the cell type mediation that is selected from mucous membrane type mast cell, serous coat type mast cell and/or basophil born of the same parents, described method is included as needs the effectively pharmaceutical composition of amount of its curee administering therapeutic, and described pharmaceutical composition contains the peptide that is selected from SEQ ID NO:3 to SEQ ID NO:8 as activating agent. In certain embodiments, the allergic disease that causes for the secretory reaction of being induced by the hypersensitivity (I type or III type) of IgE or IgG mediation and/or Fc ε RI or Fc γ R of described disease.

The example of allergic disease that can be by medicine composite for curing of the present invention includes but not limited to: allergic rhinitis comprises seasonal rhinitis and nasosinusitis; PUD D, for example bronchus asthma; Allergic skin disease, for example nettle rash, blood vessel oedema, eczema, idiocrasy dermatitis and contact dermatitis; Allergic conjunctivitis; Stomach and intestine allergy, for example the stomach and intestine allergy that causes of food or medicine; Spasm; Feel sick; Vomiting; Diarrhoea; The irritability enteropathy; The eye allergy, scorching such as eye grape film; Cheilitis; Vulvitis; And allergic reaction. The present invention also can be used for alleviating or treating the symptom that is exposed to toxin (comprising bee venom etc.) and induces. In certain embodiment, described allergic disease is asthma.

Applicant of the present invention is at United States Patent (USP) 6,682, the hypersensitivity that wherein relates to mucous membrane type mast cell (I type) that peptide with the listed amino acid sequence of SEQ ID NO:15 to SEQ ID NO:31 can be used for suppressing the IgE mediation was disclosed in 740 (its content is incorporated into by reference, is described in this as complete). But the serous coat type mast cell that these peptides present demonstration establishment IgE or IgG and/or Fc ε RI-or Fc γ R induce and basophil born of the same parents' secretory reaction.

Therefore, the invention still further relates to treatment by the method for the allergic disease of the cell type mediation that is selected from serous coat type mast cell and basophil born of the same parents, described method is included as needs the effectively peptide of at least a SEQ of being selected from ID NO:15 to the SEQ ID NO:31 of amount of its curee administering therapeutic.

In one embodiment, the peptide that is used for the treatment of the method for the allergic disease that wherein relates to serous coat type mast cell and/or basophil born of the same parents is SEQ ID NO:25 sequence:

The listed this paper of Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg is called as the peptide of C3a7, or its similar thing, derivative or salt, and described peptide is the 9-mer peptide of the 56-64 sequence of derived from human complement C 3 peptide.

In another embodiment, the peptide that is used for the treatment of the method for the allergic disease that wherein relates to serous coat type mast cell and/or basophil born of the same parents is SEQ ID NO:26 sequence:

The listed this paper of Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Arg is called as the peptide of C3a9, or its similar thing, derivative or salt, and described peptide is the g-mer peptide of the 55-62 sequence of derived from human complement PEPC 3a.

In another embodiment, the peptide that is used for the treatment of the method for the allergic disease that wherein relates to serous coat type mast cell and/or basophil born of the same parents is SEQ ID NO:27 sequence:

The listed this paper of Asp-Ser-Ser-Asn-Tyr-Ile-Arg is called as the peptide of C3a11, or its similar thing, derivative or salt, and described peptide is the similar thing of 7-mer of the 55-62 sequence of derived from human complement C 3 peptide.

In other embodiments, the peptide of the present invention that uses in the method that is used for the treatment of the allergic disease that wherein relates to serous coat type mast cell and/or basophil born of the same parents is 14-mer C3a4,20-mer C3a5,15-mer C3a6,10-mer C3a10 or its similar thing, derivative or the salt of following sequence:

C3a4：Lys-Val-Phe-Leu-Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg (SEQ ID NO：28)；

C3a5：

Lys-Lys-Val-Phe-Leu-Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg-Arg-Gln-H is-Ala-Arg(SEQ ID NO：29)；

C3a6：Lys-Val-Phe-Leu-Asp-Ala-Ala-Asn-Tyr-Ile-Thr-Glu-Leu-Arg-Arg (SEQ ID NO：30)；

C3a10：Arg-Arg-Cys-Cys-Asn-Tyr-Ile-Thr-Arg-Arg(SEQ ID NO：31)。

In certain preferred aspects, the carboxyl end of the arbitrary peptide of SEQ ID NO:25 to SEQ ID NO:31 is carboxyl end acid amides.

For the treatment of Hay Fever, for example, the pharmaceutical composition of spray or aerosol form can be suitable for delivering medicine to the curee of needs with allergic generation in the prevention pollen season. In addition, known bronchus mucomembranous surface is allergenic the first contact position that sucks, and therefore, mast cell is for very effective as replying of the inhibitory peptide of the present invention of spray administration.

The allergic disease relevant with serous coat type mast cells activation includes but not limited to I type or III type immediate hypersensitivity, for example stomach and intestine allergy, spasm, feel sick, vomiting and diarrhoea.

Peptide of the present invention can be used as monotherapy or associating other treatment agent (for example other anti-inflammatory agent) administration as pharmaceutical composition.Combination therapy can relate to conduct at the single dose form of identical time or different time administration or the drug administration of multiple doses form.

Now the present invention is described by following non-limiting examples.

Embodiment

Materials and methods

Reagent and cell culture medium.Tissue culture medium (TCM) and additive buy in Invitrogen LifeTechnologies or Gibco (Grand Island, NY).Triton X-100, p-nitrophenyl-N-ethanoyl-β-D-glucosamine and anti--Tyrosine O-phosphate Ab PT-66 from Sigma (Sigma-Aldrich Kft, Hungary).2,4-dinitrobenzene sulfonic acid link coupled bovine serum albumin (DNP11-BSA), the pearl of DNP bag quilt and mouse DNP monoclonal antibody specific A2 IgE are by Arieh Mr. Licht (Rehovot, Israel) generous present.The anti-rabbit igg of anti-mouse IgG of horseradish peroxidase (HRPO) link coupled and HRP mark is available from Sigma-Aldrich, and anti-LynAb is from BD Transduction Laboratories.Enhanced chemiluminescence reagent (ECL) is available from AmershamBiosciences (UK), and the material that is used for the SDS-gel electrophoresis is from Bio-Rad (CA, USA) acquisition.The Fluo-3AM dyestuff obtains from Calbiochem.C3a and C5a (referring to Erdei etc., Int.Immunol.7:1433-1439,1995) as discussed previously separate.

Synthetic peptide and protein labeling.Peptide is synthetic to carry out on mbha resin by the solid phase technique that uses " Boc chemistry " (Merifield etc., Biochem.14:1385-1390,1964).By reversed-phase HPLC and mass spectroscopy purifying and identify peptide.(NJ, the specification sheets that USA) provides (tagging scheme of 0.1M NaHCO3) uses Cy5 mark peptide C3a9 and uses Cy3 mark A2IgE according to Amersham-Pharmacia.

Cell.The mastocyte of bone marrow derived (BMMC) is prepared by the Balb/c mouse as (Science 212:333-335,1981) such as Nagao are described.After 3 weeks, obtain the mastocyte group of about 95% purity, measure, show the high expression level (c-kit) of Fc ε RI and STEM CELL FACTOR acceptor by flow cytometry.

From Reuben doctor Siraganian, NIH, the RBL-2H3 clone that Bethesda MD place obtains is maintained at the basic medium (DMEM) that adds 5%FCS, the antibiotic Du Beikeshi improvement of 2nM glutamine box in the wet atmosphere of 37 ℃ of 5%CO2.For experiment, by being used in the 10mM EDTA incubation 15min among the DMEM, harvested cell after separation.

(Kim etc., J.Immol.162:4960-4965,1981) as discussed previously separate rat peritoneum mastocyte (RPMC).As what assessed by the fluidic cell metering monitoring of Fc ε RI surface expression, the preparation of mastocyte is about 95% purity.The specificity activator compound 48/80 of serous coat type mastocyte cause about 50% the release of total Hex content of isolating rat peritoneum mastocyte.

The secretory reaction of mastocyte.As discussed previously (referring to Erdei etc., Int.Immunol.7:1433-1439,1995), monitor mastocyte by the activity of measuring excretory granzyme Hex and reply the medium secretion that Fc ε RI accumulative stimulates.For studying the effect of C3a and derivative thereof to the reaction of antigen inductive, with use saturation concentration DNP specificity A2 IgE sensitization mastocyte at room temperature with the different peptide preincubation 5min of finite concentration scope, be exposed to time good antigen concentration (5ng/ml) then.

Measure TNF-α by ELISA.Use rat TNF-α ELISA test kit (R﹠amp; D systems UK) is determined at and does not have or have that RBL-2H3 replys Fc ε RI accumulative TNF-α secretion in the situation of described peptide.

Immunoprecipitation and Western trace.In the 10cm culture dish, inoculate mastocyte (7 * 10 ⁶/ culture dish), in DMEM, use that A2IgE is saturated to spend the night and at 37 ℃.After the washing, use peptide that they are handled 5min, and by with 5ng/ml DNP111-BSA incubation 2min Fc ε RI being assembled at 37 ℃.By every culture dish 500 μ l cracking buffer reagents (1%Triton X-100,50mMHEPES, 100mM NaF, 10mM EDTA, the 2mM sodium orthovanadate, 10% glycerol, the 10mM trisodium phosphate, proteinase inhibitor cocktail 1: 200 pH7.4) stops this reaction.Smudge cells, and the protein content that will remove the supernatant liquor (post nuclear supernatants) behind the nucleus transfers to equivalence, the pearl precipitation (each sample 10 μ l pearl) by PT-66 Tyrosine O-phosphate specific antibody bag quilt then.Use contains the sample buffer reagent diluted protein of 2 mercapto ethanol, and uses SDS-PAGE to separate, and electrotransfer and uses specified antibody to develop to nitrocellulose filter, and detects by ECL.

Monitoring free kytoplasm Ca ²⁺Ionic concn.Be totally 5 * 10 in the 0.5ml RPMI-1640 substratum ⁶The cell of IgE sensitization loads 5 μ M Fluo-3/AM indicator and 30 μ g/ml PluronicF-127, and 37 ℃ continue 30 minutes.After the washing, with cell (5 * 10 ⁵) with the C3a7 of 200 μ M or C3a9 peptide at 37 ℃ of incubation 5min.In 20 seconds after beginning fluidic cell metering record, (DNP11-BSA) is added into cell with 5ng/ml antigen, and then with the variation of the temporal resolution mode monitoring free calcium ion concentration of Becton-DickinsonFACSCalibur flow cytometer.(NJ USA) carries out data and obtains and analyze for Becton-Dickinson, Franklin Lakes to use CELLQuest software.

Laser scanning co-focusing microscope.Results RBL-2H3 cell, and with the Cy3 link coupled IgE of 5 μ M or the Cy5 link coupled C3a9 peptide that also uses 200 μ M simultaneously at 40 ℃ of incubation 25min.After the washing, use 2% Paraformaldehyde 96, be fixed in then on the cover glass that uses 0.1% PLL precoating at fixed cell 20min on ice.In the green (exciting) of Zeiss LSM5 laser scanning confocal microscope and red (exciting) optical channel, analyze the IgE of Cy3 mark and the fluorescent signal of Cy5 peptide by 632nm He-Ne laser apparatus by 543nm He-Ne laser apparatus.Optically cell is cut into the section of 512 * 512 pixels of 0.5 μ m thickness.Carry out as the assessment of locating cross-correlation coefficient between the fluorescence intensity of measuring altogether (Vereb etc., Proc.Natl.Acad.Sci.USA 97:6013-6018,2000) as discussed previously.

Fluidic cell metering FRET (fluorescence resonance energy transfer) (FRET) is measured.Measure all and the FRET between the IgE (acceptor) of the C3a9 peptide (donor) of RBL-2H3 cell surface bonded FITC mark and Cy3 mark, and use Becton Dickinson FACSStar Plus flow cytometer is assessed.When the FITC-C3a9 peptide is 150 μ M (promptly to functional study in those similar dosage of using), use the Cy3-IgE of saturation concentration that the RBL-2H3 cell is carried out mark.

The covalent cross-linking of C3a and BMMC.The washing BMMC and at room temperature with C3a (every 15-20 * 10 ⁶Cell/sample 50 μ g/ml are in PBS) incubation 10min.At room temperature with two (sulfosuccinimide the base)-suberates of 5mM linking agent (BS3, PIERCE, IL, USA) again behind the incubation 20min, washed cell and with its cracking.(Behringwerke AG Germany) is used for immunoprecipitation to the specific multi-clone rabbit antibody of C3a.(USA) the Fc ε RI β chain specific antibody that provides of generosity carries out the Western trace for HarvardUniversity, Boston by Jean Pierre Kinet.(DAKO, Frank Diagnosztika Kft is Hungary) as second antibody to use HRPO link coupled goat anti-mouse IgG.Detect by ECL.

Surface plasma body resonant vibration (SPR) is measured.(Pharmacia Sweden) carries out SPR and measures to use 2000 type BIACORE instruments.The peptide synthetic and application has following sequence: the 1st extracellular loop that is respectively (1) rat and people Fc ε RI β chain: STLQTSDFDDEVLLL YRAGYPF (SEQ IDNO:39) and SVLDISHIEGDIFSSFKAGY (SEQ ID NO:40); And the 2nd extracellular loop of (2) rat and people Fc ε RI β chain: NNSAYMNYCKDITEDDGCFVTS (SEQ IDNO:41) and KSLAYIHIHSCQKFFETKCFMAS (SEQ ID NO:42).

All peptides are carried out biotinylation at the amino place of its N end, and with the low density of scope between 30 and 60 resonance units (RU) be connected in Streptavidin bag quilt sensing chip (BIACORE, Sweden).Be dissolved in C3a and the C5a solution in distilled water of the flow velocity of 20 μ l/min injection scope at 5 different concns of 10.5nM to 656nM.Between measuring, each use 0.1M HCl to make chip surface regeneration.Use BIAEVALUATION3.0 software analysis data.Viewed association rate constant (kob) is depicted as the function of C3a concentration, and the gradient of each curve is as specific association rate constant (kon), and the y intercept is calculated as koff/kon with Kb then as dissociation rate constant (koff).

Passive systemic anaphylaxis.Use 300-400 μ l avertin anesthesia mouse (Balb/c), and (SPE-7 is Sigma) by (or tail vein) injection behind the socket of the eye to be used in 3 μ g IgE class DNP monoclonal antibody specifics among the 200 μ l PBS.Behind the 24h, use 300-400 μ l avertin anesthesia mouse, and be exposed to the solution of the about 100 μ l specified polypeptides in PBS (inhibitor, SEQ ID NO:11, the GAKDGNEYI-COOH peptide of the contrast retropeptide of SEQ ID NO:35 or SEQ ID NO:36) that are positioned over the nostril.Then by (tail vein) injections of antigens (100 μ g are in 200 μ l PBS for DNP deutero-human serum albumin, HSA) behind the socket of the eye or only inject PBS.1.5min afterwards, put to death mouse by implementing neck dislocation and cardiac puncture.Now the syringe with the heparin flushing is used for suction.In desk centrifuge (tabletop), rotate blood sample 10min in 4 ℃ with 8000rpm.Measure histamine by the immunological technique scheme that competitive ALP measures.

Described peptide is to the inhibition effect of pulmonary function.At first, abdominal cavity (ip) injection by antigen (Ag) Protalbinic acid is carried out sensitization to mouse (Balb/c, female, 8 ages in week).Afterwards, in 1 week of every interval, attack mouse 4 times, about at every turn 20min by the aerosol suction of atomizer identical Ag of dispersive in the synthetic glass chamber.At the 28th day, at first use to be dissolved in 0.1M NaHCO ₃Dispersive is tried the aerosol processing mouse of peptide as mentioned above.Therefore, animal sucks aerosol about 10 to 20min.After the suction, attack animal by sucking (as above) sensitizing antigen (5%OVA in PBS) 20min immediately.When this processing finishes, use the pulmonary function of plethysmography test mouse under clear-headed free movement state immediately.

By enhanced in the mouse exhale intermittently (enhanced pause) monitor the bronchoconstriction degree and with the dependency of Raw air way resistance, impedance and intrapleural pressure.Ice the salt solution (x2) of precooling and extract bronchoalveolar lavage (BAL) liquid subsequently out by trachea cannula, injection 0.8ml, obtain the BAL sample from these mouse.After these tests, use Methohexitone (1mg/ml) general anesthesia to put to death mouse.Open their wall of the chest, extract blood, use cold PBS lavation lung, and carry out cytology and histological examination.

Embodiment 1: the peptide of derived from human C3a

Table 1, as follows, the form of synthetic peptide, its aminoacid sequence, mass-spectrometric data and name code is provided.Peptide C3a1, C3a3, C3a55 and GAK peptide (SEQ ID NO:33 to SEQID NO:36) are as control peptide.Use the synthetic peptide of listing in the table 1 of this paper above-mentioned " Boc chemistry ".In addition, all peptides further are prepared as amidated peptide.Should notice that the peptide synthetic method is not meant to restriction.

Embodiment 2: described peptide is to the inhibition ability of the secretory reaction of mucosal pattern mastocyte

In the experiment, the present inventor has identified the C3a sequence motifs (Erdei etc., Immunol.Lett.68:79-82,1999) of the stimulation that the IgE of responsible inhibition RBL-2H3 cell mediates in early days.The result has proved clearly that the C terminal sequence (known is vital) of C3a (65-77 position residue) is irrelevant with described inhibition in the irritated toxicity of bringing into play the complement peptide and chemically reactive.Yet, relate to and comprise the 56-64 position residue upstream sequence of (CCNYITELR is called as C3a7).Several analogues that now synthesize this sequence, wherein hot peptide DCCNYITR (being called as C3a9) show the secretion (Fig. 1) of the mucosal pattern mastocyte of the RBL-2H3 system that can effectively suppress Fc ε RI mediation.With the cell and the described peptide incubation 5min of IgE sensitization, then, use time good antigen dose (5ng/ml) to stimulate, then measure the activity of excretory granzyme Hex.Figure 1A has shown the dose-dependent inhibition of these peptides to these cell response performances.

The peptide that is called as C3a31, C3a32 and C3a35 is shown in Figure 1B to the effect of mucosal pattern mastocyte secretory reaction.Shown in Figure 1B, find that C3a31 and C3a32 peptide have suppressed the Hex secretion of the RBL-2H3 of IgE mediation.Detected C3a11 and C3a13 peptide, their demonstrations suppress the IgE dependency of RBL-2H3 cell and take off particle.

Use the mastocyte (BMMC) of bone marrow derived to carry out similar experiment.Find that C3a can suppress the secretory reaction (table 2) of these mucosal pattern mastocyte of IgE mediation, and not effect of C5a.As shown in table 2, C3a7 and C3a9 peptide are suitable with the restraining effect that the RBL-2H3 cell is brought into play to the inhibition effect of BMMC secretory reaction.The control peptide of sequence D VSNYITR does not all have effect to any system.

Table 2.C3a and C3a deutero-peptide are to RBL-2H3, the BMMC of IgE mediation and the degranulated restraining effect (IC50, μ M) of rat peritoneum mastocyte (RPMC)

Measure for taking off particle, used time good antigen dose.

^*C3a when being added into non-sensitized cell, only stimulates serous coat type RPMC, but not stimulating mucosal type RBL-2H3 and BMM cell.

Embodiment 3: described peptide is to the inhibition ability of the secretory reaction of serous coat type mastocyte

Although the mucosal pattern mastocyte stimulates no response to Toplink, known for a long time serous coat type mastocyte is stimulated by the positively charged ion succagoga.Irritated toxicity peptide C3a and C5a are by starting the emiocytosis reaction with the mast cell-expressed receptors bind of its serous coat type separately.In order to study of the effect of described peptide, use RPMC to this mastocyte type.As expected, these cells are not all replied (table 2) to C5a and C3a.Yet, when with peptide C3a7 or C3a9 is added into the RPMC of IgE sensitization and when using time good antigen dose to stimulate behind 5min, take off particle and be suppressed (table 2).

Embodiment 4: peptide C3a7 or C3a9 suppress the TNF-α secretion of Fc ε RI aggregation inducing

Secrete the effect of described peptide of measuring by detecting TNF-α to the mastocyte late phase response.For this purpose, do not have or exist stimulate RBL-2H3 under the situation of peptide C3a7 or C3a9 after 24h, get supernatant liquor, and by ELISA detection excretory cytokine concentration.Shown in Fig. 1 C, the release of two kinds of equal these inflammatory cytokines of dose-dependent inhibition of peptide.When independent interpolation, described peptide does not have effect.

Embodiment 5: inhibitory peptide suppresses the tyrosine phosphorylation of the β subunit of Lyn, PI-3K and Fc ε RI

Fig. 2 A shows that when the RBL-2H3 cell was exposed to 200 μ M C3a7 (swimming lane 3) or C3a9 (swimming lane 4) before antigenic stimulation, the protein tyrosine phosphatase of several intracellular proteins of Fc ε RI aggregation inducing strengthened significantly and reduces.Based on this result and early stage discovery (Fig. 1), studied Fc ε RI proximal event.Known these comprise the phosphorylation of the PTK Lyn of src family to the ITAM of Fc ε RI β and γ subunit.Shown in Fig. 2 B, when being subjected to antigenic stimulation under there is the situation of C3a7 or C3a9 in cell, the phosphorylation of Lyn significantly reduces, and control peptide does not have effect.Also detected the phosphorylation of Fc ε RI β chain, found to reduce (Fig. 2 B) with respect to the phosphorylation of the cell that handled by control peptide.Find also that when being exposed to peptide C3a7 and C3a9 the tyrosine phosphorylation of PI-3K (regulating the phosphatidyl inositide phosphorylation level therefore also is the enzyme of the coupling element of key) reduces.These peptides self can not cause any change of the tyrosine phosphorylated proteins pattern of RBL-2H3 cell.

Also use rat mucosal pattern RBL-2H3 system to study the inhibition effect of peptide C3a31 to Fc ε RI coupling cascade.These result of experiment show peptide C3a31 have suppressed the phosphorylation of protein tyrosine kinase Lyn and Fc ε RI β chain.Observe inhibition after the 1min, observe inhibition after the 3min PAG phosphorylation (PAG has vital role in the adjusting of src family kinase) to the Lyn phosphorylation.Yet, also show 3min C3a31 enhancing afterwards Lyn phosphorylation.In addition, after the 5min, C3a31 suppresses the Dok-1 phosphorylation, but no effect after the 8min.It also suppresses the phosphorylation of PLC γ-2.

Embodiment 6: described peptide suppresses the [Ca of antigen induction ²⁺] _iInstantaneous increase

Because the instantaneous increase of calcium ion is one of inductive very early time incident in the cell of antigen activates, has detected C3a7 and the C3a9 effect to this process in the RBL-2H3 cell.As shown in Figure 3, as peptide C3a7 or C3a9 when 5min is added into cell before antigen is attacked, this process is subjected to remarkable inhibition.Be appointed as [the Ca that in the ion of extracellular medium, flows ²⁺] _iThe initial level that increases fast and continue to increase subsequently is suppressed.In contrast, in the situation that described peptide adds separately, no effect.

Embodiment 7:C3a is in conjunction with Fc ε RI β chain

The covalent cross-linking of C3a and BMMC.Owing on the mucosal pattern mastocyte, can not detect C3a receptor, checked described peptide to bring into play the possibility of its inhibition effect by interacting with different cell membrane components (may be 1 type Fc epsilon receptor).In the experiment, the present inventor observes C3a and does not disturb interaction between IgE and the Fc ε RI in early days, therefore, infers it and does not interact with the α chain of tetramer Fc ε RI mixture.Owing to having reported in early days that the β chain that has ITAM works as the amplification agent that Fc ε RI reacts, and has detected the interaction of this membranin on C3a and the BMMC.With cell and C3a incubation, add covalent crosslinking agent BS then ³Reagent.Then, carry out lysis and use polyclonal C3a specific antibody to carry out immunoprecipitation.Use Fc ε RI β chain specific antibody to analyze by SDS-PAGE protein isolate sample and by the Western trace.Shown in Fig. 4 A, only observe single albumen (swimming lane 1), and in control sample, do not tell any albumen (swimming lane 2).Because the mixture of covalent cross-linking is to the specific reaction of each component, promptly C3a is in a side, and the β chain is at opposite side, and the unique band with 45kDa quality that occurs in swimming lane 1 is the covalent complex (9kDa) of C3a and β chain (36kDa).The result that these results and we use the RBL-2H3 cell to obtain in early days (Erdei etc., 1999, the same) in full accord.

Interactional surface plasma body resonant vibration (SPR) between C3a and the Fc ε RI β chain is measured.Be the interaction of further studying the β chain of C3a and Fc ε RI, use the immobilization oligopeptides of the first and second extracellular loop sequences to carry out the SPR measurement with people and rat tetratransmembrane (tetraspan) molecule.People C3a and C5a in contrast are as analyte.Shown in Fig. 4 B and C, C3a combines with first extracellular loop of people and rat protein with the Kd value of 250nM and 520nM respectively.Do not detect the interaction between second extracellular loop of C3a and epicyte protein.The sequence of any detection of C5a and Fc ε RI β chain does not react.

Embodiment 8:C3a9 and Fc ε RI bonded IgE are positioned on the complete RBL-2H3 cell altogether

For observe on the intact cell C3a deutero-peptide and in conjunction with the details of the spatial relation between the IgE of Fc ε RI, use Cy3 link coupled IgE and Cy5 link coupled C3a9 peptide and RBL-2H3 is carried out confocal laser scanning microscope, CLSM measure.Simple crosscorrelation between transmitting for Cy3 and Cy5 (cross-correlation coefficient: showing 〉=0.54) by pixel analysis, locate highly altogether with the IgE that combines Fc ε RI alpha subunit in conjunction with the peptide of cell.This is consistent with every other discovery, and the interaction sites of having supported described peptide strongly is in the Fc ε RI of many subunits of mastocyte receptor complex (Fig. 4 D).In addition, also detect the person monocytic cell who lacks the β chain, do not find detectable peptide combination by copolymerization collection microscopy.

The above results further obtains showing the support of the FRET measuring result of nano level adjacency.Fluidic cell metering resonance energy shifts (FCET) efficient histogram (Fig. 4 E) and shows average 24% FRET efficient, clearly shows the FITC-C3a9 peptide and is bonded to molecule adjacency between the Cy3-IgE of RBL-2H3 cell surface.

Embodiment 9: the inhibition of passive systemic anaphylaxis

Detected of the effect of C3a deutero-peptide to the mouse passive systemic anaphylaxis.As shown in Figure 5, find that the C3a31 acid amides alleviates the symptom of passive systemic anaphylaxis, monitors as the histamine levels of measuring in the mouse blood.The retropeptide that is called as the C3a55 acid amides invalid (Fig. 5).Use has the irrelevant peptide of the aminoacid sequence GAKDGNEYI-COOH of SEQ IDNO:36 and handles control group, injections of antigens DNP-HSA then.

Embodiment 10: the inhibition ability of C3a deutero-peptide in the asthmatic model

As test part and describe, attacked four times mouse (or the mouse of non-exposure, promptly be used for the mouse of testing first) mensuration pulmonary function then by being exposed to this antigenic aerosol by the sensitization of initial antigen (Protalbinic acid) injection being exposed to.At last mouse is exposed to the only aerosol of arbitrary buffer reagent, or is exposed to the buffer reagent that contains peptide C3a31 or is exposed to the buffer reagent that contains the retropeptide that is called as C3a55, attacked by antigenic final (the 5th usually).Fig. 6 A-D has described the protective capability of peptide C3a31.Fig. 6 A has described each numerical value of the lung airway resistance that is used to the mouse of testing first; Fig. 6 B has described each numerical value of the lung airway resistance of asthma mouse; Fig. 6 C has described each numerical value of the lung airway resistance of " asthma " mouse of handling with peptide C3a31 before Ag (Protalbinic acid) aerosol challenge; Fig. 6 D has described each numerical value of the lung airway resistance of " asthma " mouse of handling with peptide C3a55 before Ag (Protalbinic acid) aerosol challenge; Fig. 6 E has shown mean value and the standard deviation of result shown in Fig. 6 A-D.These results have shown that clearly the C3a31 peptide can effectively alleviate the symptom of the antigen induction of animal model in asthma.

Table 3 provides the analytical results of cell in the bronchoalveolar lavage thing (BAL) that is present in the mouse of handling by the scheme identical with those mouse of standing analysis of Pulmonary Function.As shown in table 3, use peptide C3a31 to handle detected cell type among the remarkably influenced BAL, mainly reduce the quantity of neutrophilic granulocyte.

Table 3: be exposed to of the influence of the aerosol of described peptide to the cell distribution in the bronchoalveolar lavage thing (BAL)

	Be used to the mouse of testing first	Protalbinic acid (OVA)	Protalbinic acid+peptide (OVA+Pep) (1)
				Neutrophilic granulocyte	1.7％	19％	13％
Eosinocyte
		0	25％	4％
Lymphocyte					1.3％	8％	81％
	Scavenger cell	97％	47％	1％
Mastocyte
		0	0	4％

Result of the present invention determines that clearly C3a deutero-peptide suppresses the related indication remarkable ability of transformation reactions.This observes in the cell culture of checking mucosal pattern and serous coat type mastocyte and in the animal body inner model of the similar remarkable inhibition that shows symptoms such as asthma.

It will be understood by those skilled in the art that the present invention be not subjected to above special shown in and described those content constraints.And scope of the present invention is limited by claim hereinafter.

Sequence table

＜110〉Yeda Research and Development Co., Ltd.

＜120〉complement C 3 deutero-peptide and uses thereof

＜130〉Ye Da/059 PCT

<150>US 60/702,627

<151>2005-07-27

<150>US 60/776,668

<151>2006-02-27

<160>42

＜170〉PatentIn 3.3 versions

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Ser Val Gln Leu Thr Glu Lys Arg Met Asp Lys Val Gly Lys Tyr Pro

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Lys Glu Leu Arg Lys Cys Cys Glu Asp Gly Met Arg Glu Asn Pro Met

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Arg Phe Ser Cys Gln Arg Arg Thr Arg Phe Ile Ser Leu Gly Glu Ala

35 40 45

Cys Lys Lys Val Phe Leu Asp Cys Cys Asn Tyr Ile Thr Glu Leu Arg

50 55 60

Arg Gln His Ala Arg Ala Ser His Leu Gly Leu Ala Arg

65 70 75

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When Xaa did not exist, the Asp on 2 can use low-grade alkane acidyl to modify

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Xaa Xaa Asp Ser Ser Asn Tyr Ile Thr Arg

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<222>(4)..(4)

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Xaa Cys Asn Xaa Ile Thr Xaa Leu Xaa

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<220>

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Arg-NH2 or Agm

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Xaa Cys Asn Xaa Ile Thr Arg Xaa

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Asp Cys Cys Asn Xaa Ile Thr Xaa Leu Xaa

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Asp Cys Cys Asn Xaa Ile Thr Arg Xaa

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Arg Arg Cys Cys Asn Xaa Ile Thr Xaa Leu Xaa

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Xaa Lys Val Phe Leu Asp Ala Ala Asn Xaa Ile Thr Xaa Leu Xaa Xaa

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<220>

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Xaa Lys Val Phe Leu Asp Cys Cys Asn Xaa Ile Thr Xaa Leu Xaa

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<222>(10)..(10)

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Xaa Lys Val Phe Leu Asp Cys Cys Asn Xaa Ile Thr Xaa Leu Xaa Xaa

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Gln His Xaa Xaa

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Xaa Asp Ser Ser Asn Tyr Ile Xaa

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Ser Val Gln Leu Thr Glu Lys Arg Met Asp Lys Val Gly Lys Tyr Pro

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Arg Gln His Ala Arg Ala Ser His Leu Gly Leu Ala Arg

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Arg Thr Ile Tyr Asn Ser Ser Asp Ser

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Gly Ala Lys Asp Gly Asn Glu Tyr Ile

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Lys Ser Leu Ala Tyr Ile His Ile His Ser Cys Gln Lys Phe Phe Glu

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20

Claims (24)

1. peptide or its analogue, chemical derivative or the pharmacologically acceptable salts of the 55-64 amino acids sequence of a derived from human complement C 3 (SEQ ID NO:2), described peptide has the aminoacid sequence of following general formula I:

X1-Asp-X2-Asn-Tyr-Ile-Thr-X3 (SEQ ID NO:3 to 10),

Wherein

X1 is selected from hydrogen, low-grade alkane acidyl, Cys, Ser, D-Ala and D-Ala-D-Ala;

X2 is selected from Ser-Ser and Val-Val; And

X3 is selected from Arg, Arg-NH2, Glu-Cys-Arg and Glu-Cys-Arg-NH2;

Condition is: when X1 is hydrogen or low-grade alkane acidyl and X3 when being Arg or Arg-NH2, X2 is Val-Val.

2. according to the peptide of claim 1, wherein said secretory reaction is induced by the stimulation that is selected from following group: (i) triggering of IgE or IgG mediation and (ii) Fc ε RI or Fc γ R gathering.

3. according to the peptide of claim 1, described peptide has and is selected from following group aminoacid sequence:

(a)D-Ala-D-Ala-Asp-Ser-Ser-Asn-Tyr-Ile-Thr-Arg-Z(SEQ ID NO：7)；

(b)Ser-Asp-Ser-Ser-Asn-Tyr-Ile-Thr-Arg-Z(SEQ ID NO：11)；

(c)Asp-Val-Val-Asn-Tyr-Ile-Thr-Arg-Z(SEQ ID NO：12)；

(d)Cys-Asp-Ser-Ser-Asn-Tyr-Ile-Thr-Arg-Z(SEQ ID NO：13)；

(e)Ser-Asp-Ser-Ser-Asn-Tyr-Ile-Thr-Glu-Cys-Arg-Z(SEQ ID NO：14)；

(f) (a) and (b), (c), (d) or analogue (e);

(g) (a) and (b), (c), (d), (e) or chemical derivative (f); And

(h) (a) and (b), (c), (d), (e), (f) or salt (g),

Wherein Z represents to hold carboxylic acid, acid amides or alcohol.

4. according to each peptide of claim 1 to 3, wherein said Z is the carboxyl terminal acid amides.

5. according to the peptide of claim 4, described peptide has the listed aminoacid sequence of SEQ ID NO:7, and wherein Z is the carboxyl terminal acid amides.

6. according to the peptide of claim 4, described peptide has the listed aminoacid sequence of SEQ ID NO:11, and wherein Z is the carboxyl terminal acid amides.

7. pharmaceutical composition, described pharmaceutical composition contains: as promoting agent according to each peptide of claim 1 to 6, and pharmaceutically acceptable carrier.

8. according to the pharmaceutical composition of claim 7, wherein said peptide has the aminoacid sequence that is selected from SEQ ID NO:3 to SEQ ID NO:14 and analogue or chemical derivative.

9. pharmaceutical composition according to Claim 8, wherein said peptide has the listed aminoacid sequence of SEQ ID NO:7, and optional have a carboxyl terminal acid amides.

10. pharmaceutical composition according to Claim 8, wherein said peptide has the listed aminoacid sequence of SEQ ID NO:11, and optional have a carboxyl terminal acid amides.

11. method that is used to prevent and/or treat allergic disorder, described method be included as the curee who needs it use contain the treatment significant quantity according to claim 1 to 6 each peptide and the pharmaceutical composition of pharmaceutically acceptable carrier, thereby prevention or treat described allergic disorder.

12. according to the method for claim 11, wherein said peptide has the aminoacid sequence that is selected from SEQ ID NO:3 to SEQ ID NO:14, chooses wantonly to have the carboxyl terminal acid amides.

13. according to the method for claim 12, wherein said peptide has the listed aminoacid sequence of SEQ ID NO:7, chooses wantonly to have the carboxyl terminal acid amides.

14. according to the method for claim 12, wherein said peptide has the listed aminoacid sequence of SEQ ID NO:11, chooses wantonly to have the carboxyl terminal acid amides.

15. according to the method for claim 11, wherein said allergic disorder is caused by IgE or IgG mediated hypersensitivity (I type or III type) and/or Fc ε RI and/or the secretory reaction of Fc γ R inductive.

16. according to the method for claim 11, wherein said allergic disorder is by the cell type mediation that is selected from mucosal pattern mastocyte, serous coat type mastocyte and basophilic granulocyte.

17. according to the method for claim 16, wherein said allergic disorder is selected from allergic rhinitis, pulmonary disorder, allergic skin disease, allergic conjunctivitis, gastrointestinal allergy, spasm, nausea,vomiting,diarrhea, irritability enteropathy, eye transformation reactions, cheilitis, vulvitis and anaphylaxis.

18. according to the method for claim 17, wherein said pulmonary disorder is an asthma.

19. method that is used to prevent and/or treat allergic disorder, described allergic disorder is by the cell type mediation that is selected from serous coat type mastocyte and basophilic granulocyte, described method is included as the curee who needs it and uses and contain the peptide for the treatment of significant quantity and the pharmaceutical composition of analogue, chemical derivative and pharmacy acceptable salt and pharmaceutically acceptable carrier thereof, and described peptide is selected from following sequence:

(a) X1-Cys-Asn-R1-X4 (SEQ ID NO:15 to 20);

(b) X2-Lys-Val-Phe-Leu-Asp-X3 (SEQ ID NO:21 to 23); And

(c)X5-Asp-Ser-Ser-Asn-Tyr-Ile-R7(SEQ ID NO：24)；

Wherein

X1 is selected from hydrogen, low-grade alkane acidyl, Cys, Asp-Cys and Arg-Arg-Cys;

X2 is selected from hydrogen, low-grade alkane acidyl and Lys;

X3 is selected from

(i)Ala-Ala-Asn-R1-Ile-Thr-R2-Leu-R3-R4；

(ii) Cys-Cys-Asn-R1-Ile-Thr-R2-Leu-R3; And

(iii)Cys-Cys-Asn-R1-Ile-Thr-R2-Leu-R3-R4-Gln-His-R5-R6；

X4 is selected from (i) Ile-Thr-R2-Leu-R3; (ii) Ile-Thr-Arg-R7;

X5 is selected from low-grade alkane acidyl and Leu;

R1 is selected from the die aromatischen Aminosaeuren residue;

R2 is selected from Glu and Lys;

R3 is selected from positively charged amino-acid residue;

R4 is selected from Arg and Glu;

R5 is selected from Ala and Arg;

R6 is selected from Arg and Lys;

R7 is selected from hydroxyl (OH), Arg, Arg-NH2 and Agm (agmatine).

20. according to the method for claim 19, wherein said peptide is selected from following sequence:

(a)Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg(SEQ ID NO：25)；

(b)Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Arg(SEQ ID NO：26)；

(c)Asp-Ser-Ser-Asn-Tyr-Ile-Arg(SEQ ID NO：27)；

(d)Lys-Val-Phe-Leu-Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg(SEQID NO：28)；

(e)Lys-Lys-Val-Phe-Leu-Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg-Arg-Gln-His-Ala-Arg(SEQ ID NO：29)；

(f) Lys-Val-Phe-Leu-Asp-Ala-Ala-Asn-Tyr-Ile-Thr-Glu-Leu-Arg-Arg is called as C3a6 (SEQ ID NO:30) herein;

(g) Arg-Arg-Cys-Cys-Asn-Tyr-Ile-Thr-Arg-Arg is called as C3a10 (SEQ ID NO:31) herein;

(h) (a) and (b), (c), (d), (e), (f) or analogue (g);

(i) (a) and (b), (c), (d), (e), (f), (g) or chemical derivative (h);

(g) (a) and (b), (c), (d), (e), (f), (g), (h) or salt (i);

And pharmaceutically acceptable carrier.

21. according to claim 19 and 20 each methods, wherein said peptide is made up of the listed aminoacid sequence of SEQ IDNO:25, optional have a carboxyl terminal acid amides.

22. according to each method of claim 19 to 20, wherein said peptide is made up of the listed aminoacid sequence of SEQ IDNO:26, optional have a carboxyl terminal acid amides.

23. according to the method for claim 19, wherein said allergic disorder is caused by IgE or IgG mediated hypersensitivity (I type or III type) and/or Fc ε RI and/or the secretory reaction of Fc γ R inductive.

24. according to the method for claim 19, wherein said allergic disorder is selected from gastrointestinal allergy, spasm, nausea,vomiting,diarrhea, irritable bowel disease.

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