Methods of depressing inflammation mediator release and peptides applied for this purpose

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to inhibiting or decreasing a level of inflammation mediator release from inflammatory cells by suppressing a mechanism associated with inflammation mediator release from inflammatory cell granules by using the versions of MANS peptide.

EFFECT: invention refers to an endocellular signal transmission mechanism which allows detecting a number of new endocellular targets for the pharmacological treatment of disorders associated with inflammation mediator secretion from inflammatory cell vesicles.

25 cl, 15 dwg, 6 ex

 

Cross-reference to related application

In the present application claims the priority of U.S. patent reg. No. 60/833239 filed July 26, 2006 and entered in its entirety in the present description by reference.

The scope to which the invention relates

The present invention relates to peptides or peptide compositions and methods of use thereof for attenuating or inhibiting, or reducing the level of release of mediators from inflammatory cells stimulated in the process of inflammation. The present invention also relates to the use of these peptides or peptide compositions for the modulation of the mechanism of intracellular signal transduction regulating the secretion of inflammatory mediators from inflammatory cells.

Prior art

Inflammatory leukocytes synthesize a number of inflammatory mediators that are inside cells isolated and stored in cytoplasmic membrane-bound granules. Examples of such mediators include, but are not limited to, myeloperoxidase [MPO] neutrophils (see, for example, Borregaard N, Cowland, J. B. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 1997; 89:3503-3521), peroxidase eosinophils [EPO] and basically major protein [MBP] eosinophils (see, for example, Gleich G J. Mechanisms of eosinophil-associated inflammation.J. Allergy Clin. Immunol.2000; 105:651-663), lizol is in monocytes/macrophages (see, for example, Hoff T, Spencker T, Emmendoerffer A., Goppelt-Struebe M. Effects of glucocorticoids on the TPA-induced monocytic differentiation.J. Leukoc Biol. 1992; 52:173-182; and Balboa M A, Saez Y, Balsinde J. Calcium-independent phospholipase A2 is required for lysozyme secretion in U937 promonocytes.J. Immunol.2003; 170:5276-5280) and Grasim in natural cells natural killer (NK) cytotoxic lymphocytes (see, for example, Bochan MR, Goebel WS, Brahmi Z. Stably transfected antisense granzyme B and perforin constructs inhibit human granule-mediated lytic ability.Cell Immunol.1995; 164:234-239; J.H. Gong, Maki G, Klingemann H.G. Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells.Leukemia1994; 8:652-658; Maki G, Kiingemann H.G, Martinson J.A, Tarn Y.K. Factors regulating the cytotoxic activity of the human natural killer cell line, NK-92.J. Hematother Stem Cell Res. 2001; 10:369-383; and Takayama H, Trenn G, M.V. Sitkovsky A novel cytotoxic T lymphocyte activation assay.J. Immunol. Methods1987; 104:183-190). These mediators are released in the area of injury and participate in the development of inflammation and repair tissue, for example, in the lungs and in any other organs. It is known that leukocytes release these granules according to the mechanism of exocytosis (see, for example, Burgoyne R.D, Morgan A. Secretory granule exocytosis.Physiol. Rev. 2003; 83:581-632; and Logan M.R, Odemuyiwa S.O, Moqbel R. Understanding exocytosis in immune and inflammatory cells: the molecular basis of mediator secretion.J. Allergy Clin. Immunol.2003; 111:923-932), but a more accurate description of regulatory molecules and specific pathways involved in the process of exocytosis, is not given.

Some exogenous stimulants can cause degranulation of cells by a mechanism that involves an act is vazio of protein kinase C and subsequent phosphorylation events (see, for example, Burgoyne R.D, Morgan A. Secretory granule exocytosis.Physiol. Rev.2003; 83:581-632; Logan M.R, Odemuyiwa S.O, Moqbel R. Understanding exocytosis in immune and inflammatory cells: the molecular basis of mediator secretion.J. Allergy Clin. Immunol.2003; 111:923-932; Smolen J.E, Sandborg R.R. Ca2+-induced secretion by electropermeabilized human neutrophils: the roles of Ca2+, nucleotides and protein kinaseC. Biochim Biophys Acta1990; 1052:133-142; Niessen H.W, A.J. Verhoeven Role of protein phosphorylation in the degranulation of electropermeabilized human neutrophils.Biochim, Biophys. Acta1994; 1223:267-273; and Naucler C, Grinstein S, Sundler R., Tapper H. Signaling to localized degranulation in neutrophils adherent to immune complexes.J. Leukoc. Biol. 2002; 71:701-710).

Protein MARCKS (where used herein, the designation MARCKS means "monitorowanie rich in alanine substrate kinase C" ("MyristoylatedAlanine-RichCKinaseSubstrate") is a frequent target of phosphorylation of protein kinase C (PKC), and is expressed in leukocytes at a high level (see, for example, Aderem A. A., Albert K.A., Keum M.M., Wang, J.K., Greengard P Cohn Z.A. Stimulus-dependent myristoylation of a major substrate for protein kinase C.Nature1988; 332:362-364; Thelen M, Rosen A, Nairn A.C., Aderem A. Regulation by phosphorylation of reversible association of a myristoylated protein kinase C substrate with the plasma membrane.Nature1991; 351:320-322; and J.H. Hartwig, Thelen M, Rosen A, Janmey P.A, Nairn AC, Aderem A. MARCKS is an actin filament crosslinking protein regulated by protein kinase C and calcium-calmodulin.Nature1992; 356:618-622). Protein MARCKS mechanistically involved in the process ekzoticheskoy secretion of mucin goblet cells that line the respiratory tract (see, for example, Li et al., J. Biol. Chem. 2001; 276:40982-40990; and Singer et al.,at. Med.2004; 10:193-196). MARCKS myristoylated through amide bond at the N-terminal amino acids in the amino acid sequence of MARCKS protein in the regulation of alpha-amino group of glycine, which is present at the N-Terminus (position 1) amino acid sequence. In the epithelial cells of the respiratory tract monitorowania N-terminal region of MARCKS, obviously, is an integral part of the secretory process. The term "N-end of the MARCKS protein" means the MANS peptide, containing sequence of myristoyl-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1), which is a sequence consisting of L-amino acids. In addition, described herein peptide fragments of the MANS peptide also preferably consist of L-amino acids. It is obvious that this mechanism is the binding monitorowania MARCKS protein, that is, with the membranes of intracellular granules.

It was shown that N-terminal monitorowanie peptide that is present at the N-terminal MARCKS, blocks the secretion of mucin binding of MARCKS with membranes Musinovich granules in goblet cells (see, e.g., Singer et al.,Nat. Med. 2004; 10:193-196). This peptide contains 24 amino acids of the protein MARCKS starting with N-terminal glycine protein MARCKS, which is monitorowanym through amide bond and is known as monitorowania alpha N-terminal sequence (MANS), i.e. m is retail-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1). In the publication Vergeres et al.,J. Biochem.1998, 330; 5-11, it is reported that N-terminal glycine residue protein MARCKS myristoylated through reactions catalyzed by myristoyl - CoA:protein N-myristoyltransferase (NMT).

In inflammatory diseases, such as asthma, COPD and chronic bronchitis; hereditary diseases such as cystic fibrosis; allergic States (atopy, allergic inflammation); if bronchiectases and various acute infectious respiratory diseases such as pneumonia, rhinitis, flu or cold, as well as arthritis or autoimmune diseases, inflammatory cells are usually present at sites of injury or infection associated with inflammatory pathological conditions or migrate to the respiratory areas, and in particular these cells are present in the respiratory or pneumatic paths or migrate into the Airways of patients suffering from specified diseases. These inflammatory cells may play a significant role in the pathology of the disease by tissue damage caused by inflammatory mediators released from these cells. One example of such injury or destruction of tissue in chronic inflammation may serve as tissue damage in patients with cystic fibrosis, where the mediators released and the neutrophil (for example, the myeloperoxidase [MPO]), induce desquamation of epithelial tissue of the respiratory tract.

MARCKS, i.e. a protein mass of approximately 82 KD, has three evolutionary conservative region (Aderem et al,Nature1988; 332:362-364; Thelen et al.,Nature1991; 351:320-322; Hartwig et al.,Nature1992; 356:618-622; Seykora et al.,J. Biol. Chem.1996; 271:18797-18802), namely N-terminal domain of the site of phosphorylation (or PSD) and multiple domain homology 2 (MH2). The sequence of the human cDNA of the protein MARCKS known and are given in the publication Harlan et al.,J.Biol. Chem. 1991, 266:14399 (GenBank reg. No. M68956), as well as in the publication Sakai et al,Genomics1992, 14:175. These sequences are also described in the application WO 00/50062, which in its entirety is introduced into the present description by reference. N-terminal alpha-amino acid sequence containing 24 amino acid residue, together with myristic acid molecule attached via amide bond to the N-terminal glycine residue involved in the binding of MARCKS with cellular membranes (Seykora et al., J. Biol. Chem. 1996; 271:18797-18802) and probably with calmoduline (Matsubara et al., J. Biol. Chem. 2003; 278:48898-48902). The specified sequence of 24 amino acids known as the MANS peptide.

Description of the invention

Part MARCKS protein in the release of inflammatory mediators from granules of infiltrated leukocytes plays an important role in the development of inflammatory-related disease is evani all tissues and organs, including lung disease characterized by inflammation of the Airways, such as asthma, COPD and cystic fibrosis. However, the inflammation and the secretion of mucus in the respiratory tract occur in two separate and independent mechanisms (Li et al.,J. Biol. Chem.2001; 276:40982-40990; Singer et al.,Nat. Med.2004; 10:193-196). The production and secretion of mucus may be the induction of various factors, including the mediators released by inflammatory cells, however, a direct link between excess mucus and inflammation has not been determined.

In one aspect of the invention, the MANS peptide may play a role in reducing speed and/or level of release of inflammatory mediators from granules or vesicles in inflammatory leukocytes.

In another aspect of the invention, the peptides that are present on the N-end MARCKS, and especially the N-terminal sequence consisting of 24 amino acids, i.e. adjacent active peptide fragments present in the N-terminal sequence MARCKS, consisting of 1-24 amino acids and having a glycine at position 1, and N-terminal amides such fragments, such as N-terminal amides of acetic acid and/or C-terminal amides such fragments, such as C-terminal amides of ammonia, can inhibit or reduce the speed and/or level of release of mediators of inflammation inflammatory leukocytes. Such inhibi the Finance or reducing the release includes the inhibition of MARCKS-associated release of inflammatory mediators from inflammatory cells.

In another aspect of the invention, the peptides that are present on the N-end MARCKS, and especially the N-terminal sequence consisting of amino acids 1-24, i.e. adjacent active peptide fragments present in the N-terminal sequence MARCKS, consisting of 1-24 amino acids and having a glycine at position 1, and N-terminal amides such fragments, such as N-terminal amides of acetic acid and/or C-terminal amides such fragments, such as C-terminal amides of ammonia, can inhibit the speed and/or release of inflammatory mediators, such as mediators inflammation identified in this application, by suppressing the process of degranulation of inflammatory leukocytes.

In another aspect of the invention, the MANS peptide and its active fragments and active amides such fragments described here can compete with native MARCKS protein for binding to the membrane of inflammatory cells, which will lead to the attenuation (reduction or suppression) MARCKS-mediated release of inflammatory mediators from granules or vesicles containing such mediators of inflammation and are present in these inflammatory cells.

Cells leukocyte types and models, which secrete the contents of specific granules in response to induced turbolover ester activation of PKC, can be used in the us for in vitro illustrate the effectiveness of the peptides according to the invention and substituted peptides (e.g., alpha-N-amides, C-terminal amides and esters) according to the invention.

Reducing the release of membrane-bound mediators of inflammation under the action of the compounds and compositions according to the invention can be demonstrated using human leukocyte cell lines. For example, neutrophils isolated from human blood, can be used to illustrate the attenuation or inhibition of the release of myeloperoxidase (MPO). Clone 15 human promyelocytic cell line HL-60 can be used to illustrate the attenuation of the release or inhibition of the release or secretion of peroxidase eosinophils (EPO) under the action of the compounds and compositions according to the invention (see, for example, Fischkoff SA Graded increase in probability of eosinophilic differentiation of HL-60 promyelocyte leukemia cells induced by culture under alkaline conditions. Leuk Res 1988; 12:679-686; H.F. Rosenberg, Ackerman, S. J., D.G. Tenen Human eosinophil cationic protein: molecular cloning of a cytotoxin and helminthotoxin with ribonuclease activity. J. Exp. Med. 1989; 170:163-176; Tiffany H.L., Li F, H.F. Rosenberg Hyperglycosylation of eosinophil ribonucleases in a promyelocytic leukemia cell line and in differentiated peripheral blood progenitor cells. J. Leukoc. Biol. 1995; 58:49-54; and Badewa A.P., C.E. Hudson, Heiman A.S. Regulatory effects of eotaxin, eotaxin-2, and eotaxin-3 on eosinophil degranulation and superoxide anion generation. Exp. Biol. Med. 2002; 227:645-651). Cell line monocytic leukemia the U937 can be used to illustrate the attenuation of the release or inhibition of the release or secretion of lysozyme under the action of the compounds and compositions according to the invention (see, for example, Hoff T, Spencker T, Emmendoerffer A., Goppelt-Struebe M. Effects of glucocorticoids on the TPA - induced monocytic differentiation.J. Leukoc. Biol.1992; 52:173-182; Balboa, M. A., Saez Y, Balsinde J. Calcium-independent phospholipase A2 is required for lysozyme secretion in U937 promonocytes. J. Immunol. 2003; 170:5276-5280; and Sundstrom C, Nilsson K. Establishment and characterization of a human histiocytic lymphoma cell line (U-937). Int. J. Cancer 1976; 17:565-577). Lymphocytic cell line cells natural killer cells NK-92 can be used to illustrate the attenuation or inhibition of the release of granzyme under the action of the compounds and compositions according to the invention (see, for example, J.H. Gong, Maki G, Klingemann H.G. Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia 1994; 8:652-658; Maki G, Klingemann H.G., Martinson J.A., Y.K. Tam Factors regulating the cytotoxic activity of the human natural killer cell line, NK-92. J Hematother Stem Cell Res 2001; 10:369-383; and Takayama H, Trenn G, M.V. Sitkovsky A novel cytotoxic T lymphocyte activation assay. J Immunol Methods 1987; 104:183-190). In a method of inhibiting or attenuating the release of mediators of inflammationin vitro, for example, described in this application, the cells of each type pre-incubated with the peptide compound or with a peptide composition according to the invention at various concentrations, and then these cells are incubated with a stimulator of the release of inflammatory mediators, such as Herbology ether. The percentage inhibition of release of mediators of inflammation determined in relation to the release of mediator in the absence of peptide connect the deposits or peptide compositions, for example, a spectrophotometric method for determining the concentration of the released neurotransmitter.

In order to demonstrate the important value of the corresponding amino acid sequence present in the peptides according to the invention, the relative ability to inhibit or reduce the level of a mediator of inflammation released by peptide that is identical to the sequence of the protein MARCKS, consisting of 24 amino acids in the N-terminal region (i.e. the MANS peptide with monitorowanie alpha N-terminal sequence) is compared with the capacity for inhibition or reduction of the level of a mediator of inflammation released under the action of a peptide containing the same 24 amino acid residue that is present in the MANS, but compared to the order of amino acid residues in the sequence MANS, located in random (i.e. peptide RNS, also called peptide with a randomized N-terminal sequence). In the cells of each of the evaluated types of MANS peptide, but not the RNS peptide reduces the release of inflammatory mediators, depending on the concentration during the period of time from 0.5 to 3.0 hours. The results obtained suggest that the corresponding amino acid sequence present in the peptides according to izaberete the Oia, which is MARCKS protein in the prescribed manner, in particular in its N-terminal region, and more specifically a sequence of 24 amino acid residues in the N-terminal region is involved in at least one intracellular pathway of inhibition of degranulation of leukocytes.

The present invention relates to a new use of a peptide sequence consisting of 24 amino acids, and alpha N-terminal acetylated peptide sequence, monitorowania polypeptide, also known as the MANS peptide and their active fragments, where these active fragments can be selected from the group of peptides having from 4 to 23 contiguous amino acid residues of the amino acid sequence of the peptide MANS, and where these fragments can be monitorowanie N-end in that case, if they do not begin with N-terminal glycine at position 1 of SEQ ID NO:1, or where these fragments can to be acylated at the N-terminal, C2- C12-acyl groups, including the group, acylated at the N-Terminus and/or C-terminal group, amidinophenoxy group NH2.

The present invention also relates to a new method of blocking MARCKS-associated cellular secretory processes, particularly processes that are MARCKS-associated release of inflammatory mediators from vocal is positive cells, in the ways of stimulation which involved a substrate of protein kinase C (PKC) protein MARCKS and in which there is a release of content from intracellular vesicles or granules.

The present invention relates to a method of inhibiting ekzoticheskogo release of at least one mediator of inflammation of at least one inflammatory cells, where the method includes contacting at least one inflammatory cells containing at least one mediator of inflammation that is present in the intracellular vesicles, at least one peptide selected from the group consisting of MANS peptide and its described here, the active segment in the amount effective to reduce the release of mediators of inflammation inflammatory cells, compared with the level of release of mediators of inflammation inflammatory cells of the same type observed in the absence at least one peptide.

The present invention also relates to a method of inhibiting the release of at least one mediator of inflammation of at least one inflammatory cells in the tissue or body fluids of an individual, where the method includes introducing into the tissue and/or a physiological fluid of an individual, with at least one inflammatory cell, on the expectation by at least one mediator of inflammation, present in the intracellular vesicles, a therapeutically effective amount of a pharmaceutical composition containing at least one peptide selected from the group consisting of MANS peptide and its described here, the active segment in the amount therapeutically effective to reduce the release of mediators of inflammation from at least one inflammatory cells, compared with the level of release of mediators of inflammation from at least one inflammatory cells of the same type in the absence of at least one peptide. More specifically, the inhibition of release of mediators of inflammation involves blocking or reducing the release of mediators of inflammation from inflammatory cells.

More specifically, the present invention relates to a method of reducing inflammation in an individual, where the method includes the introduction of a therapeutically effective amount of the pharmaceutical composition containing peptide MANS (i.e., N-myristoyl - GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1)or its active fragment. Such an active fragment has a length at least four, and preferably at least six amino acids. Used herein, the term "active fragment" of a protein MARCKS means a fragment that effect (inhibits or reduces) mediated MARCKS protein release, n is an example of mediated protein MARCKS release of a mediator of inflammation. The active fragment can be selected from the group consisting of GAQFSKTAAKGEAAAERPGEAAV (SEQ ID NO:2); GAQFSKTAAKGEAAAERPGEAA (SEQ ID NO:4); GAQFSKTAAKGEAAAERPGEA (SEQ ID NO:7); GAQFSKTAAKGEAAAERPGE (SEQ ID NO:11); GAQFSKTAAKGEAAAERPG (SEQ ID NO:16); GAQFSKTAAKGEAAAERP (SEQ ID NO:22); GAQFSKTAAKGEAAAER (SEQ ID NO:29); GAQFSKTAAKGEAAAE (SEQ ID NO:37); GAQFSKTAAKGEAAA (SEQ ID NO:46); GAQFSKTAAKGEAA (SEQ ID NO:56); GAQFSKTAAKGEA (SEQ ID NO:67); GAQFSKTAAKGE (SEQ ID NO:79); GAQFSKTAAKG (SEQ ID NO:92); GAQFSKTAAK (SEQ ID NO:106); GAQFSKTAA (SEQ ID NO:121); GAQFSKTA (SEQ ID NO:137); GAQFSKT (SEQ ID NO:154); GAQFSK (SEQ ID NO:172); GAQFS (SEQ ID NO:191) and GAQF (SEQ ID NO:211). These peptides do not contain myristoleic group at the N-terminal amino acids, that is, either do not contain chemical groups, or contain demeritorious chemical group at the N-terminal amino acids and/or chemical group at the C-terminal amino acids, such as described here, the N-terminal acetyl group and/or C-terminal amide group. The presence of the hydrophobic N-terminal myristoleic groups in peptides MANS and in their N-terminal monitorowania fragments can increase their ability to penetrate the plasma membrane, and likely to increase their permeability to plasma membranes, and possibly to the message these peptides solubility in cells. After the introduction of hydrophobic myristoleic groups in the lipid bilayer membrane of these lipids may have a distribution coefficient or seeming constant Association up to 104M-1or unitary free energy of binding Gibbs, costall the expansion of about 8 kcal/mol (see, for example, Peitzsch, R. M., and McLaughlin, S. 1993, Binding of acylated peptidas and fatty acids to phospholipid vesicles: pertinence to myristoylated proteins. Biochemistry. 32:10436-10443), where these values are sufficient, at least in part, for distribution of the MANS peptide and monitorowania fragments of MANS peptide in the plasma membrane of cells, and the additional functional groups and their interaction in the MANS peptide (which is monitorowanym) and monitorowania fragments of MANS peptide may increase their relative ability to penetrate into the membrane. Each of these fragments may have the distribution coefficients and the affinity with respect to the membranes, which by their structure are representative. These fragments can be obtained by the methods of peptide synthesis known in the art such as solid phase peptide synthesis (see, for example, the methods described in the publications Chan, Weng C. and White, Peter D.Eds., Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press, New York, New York (2000); and Lloyd-Williams, P. et al. Chemical Approaches to the Synthesis of Peptides and Proteins (1997)), and can be purified by methods known in the art, such as liquid chromatography high pressure. The molecular mass of each peptide may be confirmed using mass spectroscopy, where each peptide has a peak with a corresponding molecular weight. The effectiveness of individual peptides and their is combinaci (for example, combinations of the 2 peptides, and combinations of the 3 peptides, and combinations of the 4 peptides) in the methods according to the invention can easily be determined without undue experimentation in accordance with the procedures described in the examples presented in this application. The preferred combination contains two peptide, with the preferred molar ratio of these peptides can be from 50:50 (1:1) to 99.99 to 0.01, where the ratio can easily be determined using the procedures described in the examples presented in this application.

Preferably, the MANS peptide or an active fragment present in pharmaceutical compositions, which can be used to block inflammation. The present invention also relates to methods of inhibiting cellular secretory process in the individual, where this method includes the introduction of therapeutically effective amounts of compounds containing the MANS peptide or an active fragment that inhibits mediator of inflammation in an individual. This introduction is usually chosen from a group consisting of local administration, parenteral administration, rectal administration, intra-lungs of administration, administration by inhalation and internalnode injection or oral administration, where the specified internal-lung introduction usually carried tlaut spray, insufflator, nebulizer with a metering valve or a nebulizer .

Introduction composition comprising inhibiting the degranulation number of MANS peptide or inhibiting the degranulation of the number of its active fragment, for example, pharmaceutical compositions of the MANS peptide or an active fragment, a person or an animal provides the contacting of the MANS peptide or an active fragment that is present at least on the site, or in a cloth or tissue, or in the layer or the layer containing a physiological fluid, with the surface of the fabric, in which there are granulocytic inflammatory cells or into which they penetrate, allowing the MANS peptide or an active fragment of contact with inflammatory granulocyte cell. In one aspect of the invention, the introduction of such compositions can be carried out at the first sign or during the first detection of inflammation or at the first sensation of inflammation by a person or animal, or when the first noticeable change in the degree of inflammation in the human or animal to reduce the inflammation that occurs in the absence of MANS peptide or an active fragment. In another aspect of the invention, such introduction can be carried out in the process of inflammation of tissue in the human or animal to weaken the level of progression of the inflammation that occurs in the absence of MANS peptide or an active fragment. Although the number and frequency of the injected dose can be determined in accordance with the clinical evaluation and depend on the type of disease or inflammation, the degree of tissue damage and the age and weight of patient, but it is assumed that the dose of the pharmaceutical composition can be re-introduced through 3-8 hours, preferably 6-8 hours after the first injection of the pharmaceutical composition.

The present invention also relates to a method of attenuating inflammation in an individual, where these methods include the introduction of a therapeutically effective amount of a compound that inhibits the MARCKS-associated release of inflammatory mediators, resulting in a release rate of at least one mediator of inflammation in the individual is reduced compared to the level observed in the absence of such treatment. Used herein, the term "attenuation"essentially means reducing the degree of inflammation. Preferably, the release of inflammatory mediators is inhibited or blocked ways described here.

In another embodiment, the present invention relates to a method of attenuating inflammation in an individual, where these methods include the introduction of a therapeutically effective amount of a compound that inhibits the MARCKS-associated wysw the release of inflammatory mediators, in the result, the degree of inflammation in the individual is reduced compared with the degree of inflammation observed in the absence of such treatment. The present invention also relates to a method of weakening or ingibirovaniya inflammation in an individual, where these methods include the introduction of a therapeutically effective amount of MANS peptide or an active fragment for the inhibition of mediator of inflammation in the area of inflammation. The term "inhibition" means reducing the secretion of inflammatory mediators. The term "full inhibition" means reducing the secretion of inflammatory mediators to zero. As mentioned above, the active segment is of length at least four, and preferably at least six amino acids. The term "process of exocytosis" means exocytosis, a process of cellular secretion or excretion, in which substances contained in vesicles present inside the cells, are released from the cell by membrane fusion of vesicles with the outer cell membrane. The term "degranulation" refers to the release of the contents of the cell pellet. The term "inhibition of degranulation" means reducing the release of inflammatory mediators contained in the granules of inflammatory cells. Thus, the number of MANS peptide and/or its active fragm the NTA, inhibiting the degranulation is the number of these peptides is sufficient to reduce the release of inflammatory mediators contained in the granules, compared with the level of release of the above mediators in the absence of such a peptide.

In the reference peptide GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1), starting from N-Terminus: in position 1 is G; in position adjacent to the position 1, which is G, that is, in position 2, there is A; at the position adjacent to the position 2, in which there is A, that is, in position 3, there is Q; and in the position adjacent to the position 3, in which there is a Q, that is, in position 4, is F; in position adjacent to the position 4, in which is F, that is, in position 5, is S; in position adjacent to the position 5, which is S, that is, in position 6, there is K; in position adjacent to the position 6, which is K, that is, in position 7, is T; position adjacent to the position 7, which is T, that is, in position 8, there is A; at the position adjacent to the position 8, in which there is A, that is, in position 9, there is A; in the position adjacent to the position 9, in which there is A, that is in position 10, there is K; in position adjacent to the position 10, in which there is a K, that is in position 11, prisutstvie the em G; in the position adjacent to the position 11, in which there is a G, that is in position 12, is E; in position adjacent to the position 12, which is E, i.e. in position 13, there is A; at the position adjacent to the position 13, in which there is A, that is in position 14, there is A; at the position adjacent to the position 14, in which there is A, that is, in the position 15, there is A; at the position adjacent to the position 15, in which there is A i.e. in position 16, is E; in position adjacent to the position 16, in which there is E, i.e. in position 17, there is R; position adjacent to the position 17, in which there is R, i.e. in position 18, is P; in position adjacent to the position 18 in which there is a P at position 19, there is G; in position adjacent to the position 19, in which there is a G, that is, in the position 20, is E; in position adjacent to the position 20, which is E, i.e. in the position 21, there is A; at the position adjacent to the position 21, in which there is A, that is, in position 22, there is A; at the position adjacent to the position 22 in which there is A, that is, at position 23, there is V; and in the position adjacent to the position 23, which is V, i.e. at position 24 is present And where the position of the C-terminal 24 is the position of the reference peptide.

"Variant" of a reference peptide or variant of the segment of the reference peptide consisting of 4-23 amino acids is a peptide that has an amino acid sequence that differs from the amino acid sequence of the reference peptide or amino acid sequence segment of the reference peptide, respectively, in at least one position of amino acids amino acid sequence of the reference peptide or segment of the reference peptide, respectively, while retaining the mucin - or mucus-inhibitory activity, which is typically in the 0.1-10 times, preferably 0.2 to 6 times, and more preferably, 0.3 to 5 times greater than the specified activity reference peptide or segment, respectively. "Variant" of the reference amino acid sequence or variant of the segment of the reference amino acid sequence consisting of 4-23 amino acid is an amino acid sequence that differs from a reference amino acid sequence or segment of the reference amino acid sequence at least one amino acid, respectively, has the amino acid sequence of a peptide which retains the mucin - or mucus-inhibitory activity of the encoded amino acid sequence specified reference PE the Chida or segment, accordingly, where this activity is usually 0.1 to 10 times, preferably 0.2 to 6 times, and more preferably, 0.3 to 5 times the activity of the indicated peptide or a segment of the reference sequence, respectively. Variant peptide with replacement or a variant amino acid sequence with the substitution may differ from the amino acid sequence (i.e. not coincide with the sequence of the reference peptide or from a reference amino acid sequence one or more amino acid substitutions introduced in the reference amino acid sequence; peptide with a deletion or a variant amino acid sequence with deletion can differ from the amino acid sequence (i.e. not coincide with the sequence of the reference peptide or from a reference amino acid sequence one or more amino acid deletions, introduced in the reference amino acid sequence; peptide with additions or variant amino acid sequence with additions may differ from the amino acid sequence (i.e. not coincide with the sequence of the reference peptide or from a reference amino acid sequence one or more amino acid additions, enter nymi in the reference sequence. Variant peptide or variant amino acid sequences may be obtained by replacing one or more amino acids (for example, replacement of at least 1, 2, 3, 4, 5, 6, 7 or 8 amino acids), introduced in the reference sequence, or it can be obtained by deletion of one or more amino acids (e.g., at least 1, 2, 3, 4, 5, 6, 7 or 8 amino acids), introduced in the reference sequence, or it can be obtained by adding one or more amino acids (for example, at least 1, 2, 3, 4, 5, 6, 7 or 8 amino acids), introduced in the reference sequence, or combinations thereof in any order. Variant peptide segment from 4-23 amino acid replacement or variant sequence segment from 4-23 amino acid replacement may differ from the reference peptide segment (i.e. not coincide with it), consisting of 4-23 amino acids, or a reference sequence consisting of 4-23 amino acids of one or more amino acid substitutions introduced in the reference sequence of amino acid segment; variant peptide segment from 4-23 amino acids with a deletion or sequence segment from 4-22 amino acids with a deletion may differ from the reference peptide segment (i.e. not coincide with him) from 5-23 amino acids or from the reference amino is islotes sequence segment from 4-23 amino acids of one or more amino acid deletions, introduced in the reference sequence of amino acid segment; and variant peptide segment from 4-23 amino acids with the addition of or a variant amino acid sequence segment of 4-23 amino acids with the addition may differ from the reference peptide segment (i.e. not coincide with him) from 4-22 amino acids or from the reference sequence from 4 to 22 amino acids of one or more amino acid additions, introduced in the reference amino acid sequence. Variant of the peptide of 4-23 amino acids or amino acid sequence of 4-23 amino acids can be obtained by replacing one or more amino acids (for example, replacement of at least 1, 2, 3, 4, 5, 6, 7 or 8 amino acids), introduced in the segment of the reference amino acid sequence of 4-23 amino acids, or it can be obtained by deletion of one or more amino acids (e.g., deletions of at least 1, 2, 3, 4, 5, 6, 7 or 8 amino acids), entered in the corresponding larger reference amino acid sequence, or it can be obtained by adding one or more amino acids (e.g., deletions of at least 1, 2, 3, 4, 5, 6, 7, 8 amino acids), entered into appropriate smaller reference amino acid sequence, or it can be half the Yong in the combined modifications. Preferably, the variant peptide or amino acid sequence differs from the reference peptide or segment of the reference peptide, or a reference amino acid sequence, or from a segment of the reference amino acid sequence, respectively, less than 10 amino acid substitutions, deletions and/or additions, more preferably, less than 8 amino acid substitutions, deletions and/or additions, even more preferably less than 6 amino acid substitutions, deletions and/or additions, even more preferably, less than 5 amino acid substitutions, deletions and/or additions, and most preferably less than 4 amino acid substitutions, deletions and/or additions. Most preferably, the variant amino acid sequence differs from the amino acid sequence of the reference peptide or a segment of the sequence by one, two or three amino acids.

The term "sequence identity", if it refers to the amino acid sequences of two peptides, means the number of positions with identical amino acids, divided by the number of amino acids in the shorter of the two compared sequences.

The term "essentially identical", if it is used when comparing amino acid sequences of two peptides or aminoxy the pilot sequences of the two peptide segments (for example, segments of the amino acid sequence of the reference peptide) means that the amino acid sequence of peptides or segments of the peptides are at least 75% identity, preferably at least 80% identity, more preferably at least 90% identity, and most preferably at least 95% identity.

Used herein, the term "peptide" includes peptides, as well as its pharmaceutically acceptable salt.

Used herein, the term "isolated" peptide means a natural peptide, which was separated or, in essence, is separated from cellular components (e.g., nucleic acids and other peptides), in an environment where it is normally present in nature, by purification, recombinant synthesis or chemical synthesis, and includes not natural peptides that were synthesized by recombinant or chemical means and which have been purified or essentially purified from cellular components, biological materials, chemical precursors or other chemicals.

In the description of the present invention uses the following three-letter and one-letter symbols of amino acids: alanine: (Ala); arginine: (Arg) R; asparagine: (Asn) N; aspartic acid (Asp) D; cysteine: (Cys); glutamine: (Gln) Q; glutamic acid (Glu) E; glycine: (Gly) G; is histidin: (His) H; isoleucine: (Ile) I: leucine: (Leu) (L); lysine (Lys) K; methionine: (Met) M; phenylalanine: (Phe) F; Proline: (Pro) P; serine: (Ser); threonine: (Thr) T; tryptophan (Trp) W; tyrosine: (Tyr) Y; valine: (Val) V. In this application are used and other three-letter symbols of amino acids, given in parentheses, namely (Hyp) - hydroxyproline, (Nle) - norleucine, (Orn) - ornithine, (Pyr) - Pyroglutamate acid and (Sar) - sarcosine. In accordance with the agreement, aminocore (or N-way) peptide is indicated from the left end of the recorded amino acid sequence of the peptide, and carboxylic (or the end) is specified from the right end of the recorded amino acid sequence. Amino acid sequence of the peptide can be written in single-letter symbols, meaning of amino acids that are covalently bound peptide amide bonds.

Active fragments of the MANS peptide can be used to prevent inflammation or reduce inflammation in the tissues of the animal, where the specified inflammation caused by inflammatory mediators. Active peptide fragments MANS can also be used to prevent destruction or reduction of the level of destruction in the tissues of the animal, where the specified defeat is caused by inflammatory mediators. An active fragment of the MANS peptide consists of at least 4 contiguous amino acids and not more than 23 contiguous linakis is from the MANS peptide (SEQ ID NO:1). In the context of the present invention, the term "active fragment" means a fragment peptides MANS, which can prevent the release or reduce the release of inflammatory mediators from inflammatory cells. This reduction in release of inflammatory mediators under the action of these active fragments can be at least 5% to 99% compared with the level observed in the presence of the reference peptide, such as peptide MANS.

Table 1 lists the amino acid sequence represented by single letter codes, together with their corresponding numbers and SEQ ID NO. Amino acid sequence of the reference peptide (peptide MANS) is presented as peptide 1. Amino acid sequences of the peptides according to the invention having the amino acid sequence consisting of 4-23 contiguous amino acids of the reference amino acid sequence presented as peptides 2-231, together with a randomized amino acid sequence N-terminal sequence (RNS), containing amino acids peptide MANS and presented as peptide 232. Amino acid sequences of representative variants of amino acid sequences described in this application, as well as peptides according to the invention is presented as peptides 233-245 and 247-251. Options item is listed here peptides should not be construed as limiting the group of peptides and are only representative examples of peptides according to the invention. Also, there is a representative reverse amino acid sequence (peptide 246) and representative conducting a randomized amino acid sequence of the peptide (peptide 232) according to the invention. Reverse and conducting a randomized amino acid sequences shown in the tables, are not considered in this application as a representative sequence.

Table 1 contains a list of the peptides according to the invention and their respective amino acid sequences and corresponding SEQ ID NOS.

Amino acid sequence of the peptide shown in table 1, can be chemically modified. For example, if the amino acid sequence of the peptide shown in table 1, chemically modified at the N-terminal amine with the formation of amide carboxylic acid, such a peptide is sometimes referred to here as a combination of signs carboxylic acid, represented by prefix, United with the number of peptide by a hyphen. For example, in the case of peptide 79, N-terminal monitorowanie peptide 79 may sometimes be called here "monitorowanym peptide 79" or "mir-peptide 79 is; N-terminal acetylated peptide 79 may sometimes be called here "acetyl-peptide 79" or "Ac-peptide 79". Cyclic variant of the peptide 79 may be referred to as "cyclic peptide 79" or CEC-peptide 79". In addition, for example, if the amino acid sequence of the peptide shown in table 1, chemically modified at the C-terminal carboxyl group such as an amine, such as ammonia, with the formation of the C-terminal amide, such a peptide is sometimes referred to here as combinations of designations of amino acid residue represented as suffix, United by a hyphen with the number of peptide. For example, the C-terminal amide peptide 79 sometimes may be referred to as "peptide-NH2". If the N-terminal amine of the peptide (e.g., peptide 79) chemically modified, for example, myristoleic group, and the C-terminal carboxyl group is chemically modified, for example, ammonium group with the formation of the above-described amide, such a peptide can sometimes be indicated by using a prefix and a suffix, such as "mir-peptide 79-NH2".

The present invention relates to peptides having amino acid sequences containing less than 24 amino acids, and amino acid sequence of related amino acid sequences of the MANS peptide (i.e. the MANS peptide is merit the Il-peptide 1, and the reference amino acid sequence of the MANS peptide of 24 amino acids is a peptide 1). The peptides according to the invention consist of amino acid sequences containing less than 24 amino acids, and may consist of 8-14, 10-12, 9-14, 9-13, 10-13, 10-14, at least 9, at least 10 amino acids or other Peptides are typically peptides with straight chains, but they can also be cyclic peptides. In addition, these peptides can be isolated peptides.

As for peptide 1 (SEQ ID NO:1), that is, the reference sequence of 24 amino acids, the segment of the reference amino acid sequence of 23 contiguous amino acids are sometimes called here 23-Merom. Similarly, the segment of the reference sequence of the 22 contiguous amino acids are sometimes called here 22-Merom; amino acid sequence of 21 amino acids is called 21-Merom; amino acid sequence of the 20 amino acids is called 20-the mayor; the amino acid sequence of 19 amino acids is called 19-Merom; amino acid sequence of 18 amino acids is called 18-Merom; amino acid sequence of 17 amino acids called 17-Merom; amino acid sequence of 16 amino acids is called a 16-Merom; amino acid sequence of 15 amino acids is called the 15-Mer is m; amino acid sequence of 14 amino acids is called a 14-Merom; the amino acid sequence of 13 amino acids called 13-Merom; amino acid sequence of 12 amino acids is called a 12-Merom; amino acid sequence of 11 amino acids called 11-Merom; amino acid sequence of 10 amino acids is called a 10-Merom; amino acid sequence of 9 amino acids called 9-Merom; the amino acid sequence of 8 amino acids is called 8-Merom; amino acid sequence of 7 amino acids is called 7-Merom; amino acid sequence of 6 amino acids is called 6-Merom; the amino acid sequence of 5 amino acid called 5-Merom, and amino acid sequence of 4 amino acid called 4-Merom. In one aspect of the invention, any of these "4-23-dimensional" amino acid sequences that are themselves peptides (sometimes referred to here H2N-peptide-COOH)may be independently chemically modified, for example, by introducing chemical modifications, which can be selected from the group consisting of (i) the formation of amide N-terminal amino group (H2N-peptide-), for example, under the action of Clor preferably C2(acetic acid) - C22-carboxylic acid; (ii) is formed by the amide I at the C-terminal carboxyl group (peptide-COOH-), for example, under the action of ammonia or primary or secondary Cl-C22-amine; and (iii) combinations thereof.

Such peptides have an amino acid sequence selected from the group consisting of (a) amino acid sequences that have 4-23 contiguous amino acids of the reference sequence peptide 1; (b) a sequence that is essentially identical to the sequence defined in (a); and (c) the variant amino acid sequence defined in (a)where the specified option is selected from the group consisting of variants with substitutions, variants with deletions, the option with the additions and combinations thereof. In some embodiments of the invention, these peptides have an amino acid sequence selected from the group consisting of: (a) amino acid sequence having 8 to 14 contiguous amino acids of the reference sequence peptide 1; (b) amino acid sequence essentially identical to the sequence defined in (a); and (c) the variant amino acid sequence defined in (a)where the specified option is selected from the group consisting of variants with substitutions, variants with deletions, the option with the additions and combinations thereof. In other embodiments of the invention, these peptides have an amino acid sequence selected from the group consisting of: (a) the amino acid posledovatel the activity, having 10-12 contiguous amino acids of the reference sequence peptide 1; (b) amino acid sequence essentially identical to the amino acid sequence defined in (a); and (c) the variant amino acid sequence defined in (a)where the specified option is selected from the group consisting of variants with substitutions, variants with deletions, the option with the additions and combinations thereof. In other embodiments of the invention, these peptides have the amino acid sequence containing at least 9, at least 10, 9-14, 9-13, 10-13, 10-14, or other related amino acids of the reference sequence peptide 1; amino acid sequence essentially identical to this sequence, or their variants, where this option is selected from the group consisting of variants with substitutions, variants with deletions, the option with the additions and combinations thereof. As explained in detail below, one or more amino acids, peptides (for example, N-terminal and/or C-terminal amino acids) can be, but not necessarily, independently chemically modified; in some embodiments of the invention, one or more amino acids of the peptide can be chemically modified, and in other embodiments of the invention, none of the amino acids of the peptide is not chemically modified. In one aspect of the invention, predpochtite is supplemented flax modification may be present at the amino group (-NH 2N-terminal amino acids of the peptide or peptide segment (where this amino group forms a peptide amide bond in the case, if it is present within the peptide sequence, and not in the N-terminal position). In another aspect of the invention, the preferred modification may be present at carboxypropyl (-COOH) C-terminal amino acids of the peptide or peptide segment (where specified carboxypropyl peptide forms an amide bond in the case, if it is present within the peptide sequence, and not in the C-terminal position). In another aspect of the invention, the preferred modification may be present as the N-terminal amino group (NH2), and the C-terminal carboxyl group (-COOH).

In some embodiments of the invention, the amino acid sequence of the peptide begins with the N-terminal amino acids of the reference sequence peptide 1. For example, such peptides may have an amino acid sequence selected from the group consisting of (a) amino acid sequences that have 4-23 contiguous amino acids of the reference sequence peptide 1, where the specified amino acid sequence begins with the N-terminal amino acids of the reference sequence (peptide 2, peptide 4, peptide 7, peptide 11, peptide 16, peptide 22, peptide 29, pept is Yes 37, peptide 46, peptide 56, peptide 67, peptide 79, peptide 92, peptide 106, peptide 121, peptide 137, 154 peptide, peptide 172, peptide 191 or peptide 211); (b) a sequence essentially similar to the amino acid sequence defined in (a); and (c) the variant amino acid sequence defined in (a). These peptides do not contain chemical groups or contain chemical group at the N-terminal glycine, which is not myristoleic group. Preferred chemical group is an acyl group, represented in the form amide linkages, such as acetyl group, or an alkyl group.

In other embodiments of the invention, the amino acid sequence of the peptide ends at the C-terminal amino acids of the reference sequence peptide 1. For example, such peptides may have an amino acid sequence selected from the group consisting of (a) amino acid sequences that have 4-23 contiguous amino acids of the reference sequence peptide 1, where the specified amino acid sequence ends at the C-terminal amino acids of the reference sequence (peptide 3, peptide 6, peptide 10, peptide 15, peptide 21, peptide 28, peptide 36, peptide 45, 55 peptide, peptide 66, peptide 78, peptide 91, peptide 105, peptide 120, peptide 136, peptide 153, peptide 171, peptide 190, peptide 210 or PE the Chida 231); (b) sequence essentially similar to the amino acid sequence defined in (a); and (c) the variant amino acid sequence defined in (a).

In other embodiments of the invention, the amino acid sequence of the peptide does not begin with N-terminal amino acids of the reference sequence peptide 1 (SEQ ID NO:1)and with the provisions of amino acids from 2 to 21 of the reference sequence peptide 1. For example, these peptides may have an amino acid sequence selected from the group consisting of (a) amino acid sequences that have 4-23 contiguous amino acids of the reference sequence peptide 1, where the specified amino acid sequence begins with any provision of amino acids from 2 to 21 of the reference sequence, where these peptides may have a length of 4-23 contiguous amino acids and can be a peptide present in the middle of the reference sequence peptide 1; (b) a sequence essentially similar to the amino acid sequence defined in (a); and (c) the variant amino acid sequence in (a). Such peptides are described in tables 1 or 2. These peptides may not contain covalently linked chemical groups or contain chemical group at the N-terminal amino acids, which is not the N-terminal is licina or amino, equivalent to N-terminal glycine amino acid sequence of SEQ ID NO:1. Preferred chemical group is an acyl group such as acetyl group or Mirandolina group, represented in the form of an amide bond, or an alkyl group.

Peptide amino acid sequences that can be used in the present invention for the inhibition of mucin hypersecretion in a mammal, and which can be used to reduce hypersecretion of mucin mammal, and methods of inhibiting mucin hypersecretion and ways to reduce hypersecretion of mucin, are amino acid sequences of the selected peptides and amino acid sequences of peptides that contain, but not necessarily, N-terminal and/or C-terminal chemically modified groups according to the invention, where these peptide amino acid sequence selected from the group consisting of a 23-Mer (i.e. peptides having a sequence of 23 amino acids): peptide 2 and peptide 3; 22-mers (i.e. peptides having a sequence of 22 amino acids): peptide 4 peptide 5 peptide 6; 21-mers (i.e. peptides having a sequence of 21 amino acids), peptide 7 peptide 8, peptide 9 and peptide 10; 20-mers (i.e. peptides having posledovatel the ability of the 20 amino acids): peptide 11, peptide 12, peptide 13, peptide 14 and peptide 15; 19-Mer (i.e. peptides having a sequence of 19 amino acids): peptide 16, peptide 17, peptide 18, peptide 19, peptide 20 and peptide 21; 18-mers (i.e. peptides having a sequence of 18 amino acids): peptide 22, peptide 23, peptide 25, peptide 26, peptide 27 and peptide 28; 17-mers (i.e. peptides having a sequence of 17 amino acids): peptide 29; peptide 30, peptide 31, peptide 32, peptide 33, peptide 34, 35 peptide and peptide 36; 16-mers (i.e. peptides having a sequence of 16 amino acids): 37 peptide, peptide 38, peptide 39, 40 peptide, peptide 41, 42 peptide, peptide 43, 44 peptide and peptide 45; 15-mers (i.e. peptides having a sequence of 15 amino acids): peptide 46, peptide 47, peptide 48, peptide 49, peptide 50, peptide 51, peptide 52, peptide 53, peptide 54 peptide 55; 14-mers (i.e. peptides having a sequence of 14 amino acids): peptide 56, 57 peptide, peptide 58, peptide 59, peptide 60, peptide 61, 62 peptide, peptide 63, peptide 64, peptide 65 and 66 peptide; 13-mers (i.e. peptides having a sequence of 13 amino acids): peptide 67, peptide 68, peptide 69, peptide 70, peptide 71, peptide 72, peptide 73, peptide 74, peptide 75, peptide 76, peptide 77 and peptide 78; 12-mers (i.e. peptides having a sequence of 12 amino acids): peptide 79, peptide 80, peptide 81, peptide 82 peptide 83, peptide 84, peptide 85, peptide 86, peptide 87, 88 peptide, peptide 89, 90 peptide and peptide 91; 11-mers (i.e. peptides having a sequence of 11 amino acids): peptide 92, peptide 93, peptide 94, peptide 95, peptide 96, peptide 97, peptide 98, 99 peptide, peptide 100, peptide 101, peptide 102, peptide 103, peptide 104 and peptide 105; 10-mers (i.e. peptides having a sequence of 10 amino acids): peptide 106, peptide 107, peptide 108, peptide 109, peptide 110, peptide 111, peptide 112, peptide 113, peptide 114, peptide 115, peptide 116, 117 peptide, peptide 118, 119 peptide and peptide 120; 9-mers (i.e. peptides having a sequence of 9 amino acids): peptide 121, peptide 122, peptide 123, peptide 124, peptide 125, 126 peptide, peptide 127, peptide 128, peptide 129, peptide 130, peptide 131, peptide 132, peptide 133, peptide 134, 135 peptide peptide 136; 8-mers (i.e. peptides having a sequence of 8 amino acids): peptide 137, peptide 138, peptide 139, 140 peptide, peptide 141, peptide 142, peptide 143, peptide 144, peptide 145, peptide 146, peptide 147, peptide 148, 149 peptide, peptide 150, 151 peptide, peptide 152 and peptide 153; 7-mers (i.e. peptides having a sequence of 7 amino acids): peptide 154, 155 peptide, peptide 156, peptide 157, peptide 158, peptide 159, peptide 160, peptide 161, 162 peptide, peptide 163, peptide 164, peptide 165, peptide 166, 167 peptide, peptide 168, peptide 169, peptide 170, pepti is and 171; 6-mers (i.e. peptides having a sequence of 6 amino acids): peptide 172, peptide 173, peptide 174, peptide 175, peptide 176, peptide 177, peptide 178, peptide 179, peptide 180, peptide 181, peptide 182, peptide 183, peptide 184, peptide 185, peptide 186, peptide 187, peptide 188, peptide 189 peptide 190; 5-mers (i.e. peptides having the sequence of 5 amino acids): peptide 191, peptide 192, peptide 193, peptide 194, peptide 195, peptide 196, peptide 197, peptide 198, peptide 199, peptide 200, peptide 201, peptide 202, peptide 203, peptide 204, peptide 205, peptide 206, peptide 207, peptide 208, peptide 209 peptide 210; and 4-mers (i.e. peptides having a sequence of 4 amino acids): peptide 211, peptide 212, peptide 213, peptide 214, peptide 215, peptide 216, 217 peptide, peptide 218, peptide 219, 220 peptide, peptide 221, peptide 222, peptide 223, peptide 224, peptide 225, peptide 226, 227 peptide, peptide 228, peptide 229, peptide 230 and peptide 231.

Preferred amino acid sequences of the selected peptides and chemically modified at the N - and/or-to the ends of the peptides according to the invention selected from the group consisting of a 23-Mer: peptide 2 and peptide 3; 22-mers: peptide 4 peptide 5 peptide 6; 21-mers: peptide 7 peptide 8, peptide 9 and peptide 10; 20-mers: peptide 11, peptide 12, peptide 13, peptide 14 and peptide 15; 19-mers: peptide 16, peptide 17, peptide 18, peptide 19, peptide 20 and the peptide is 21; 18-mers: peptide 22, peptide 23, peptide 24, peptide 25, peptide 26, peptide 27 and peptide 28; 17-mers: peptide 29, peptide 30, peptide 31, peptide 32, peptide 33, peptide 34, 35 peptide: peptide 36; 16-mers: peptide 37, peptide 38, peptide 39, 40 peptide, peptide 41, 42 peptide, peptide 43, 44 peptide and peptide 45; 15-mers: peptide 46, peptide 47, peptide 48, peptide 49, peptide 50, peptide 51, peptide 52, peptide 53 and peptide 54; 14-mers: peptide 56, 57 peptide, peptide 58, peptide 59, peptide 60, peptide 61, 62 peptide, peptide 63 peptide 64: 13-mers: peptide 67, peptide 68, peptide 69, peptide 70, peptide 71, peptide 72, peptide 73, peptide 74 and peptide 75; 12-mers: peptide 79, peptide 80, peptide 81, peptide 82, peptide 83, peptide 84, peptide 85, peptide 86 and peptide 87; 11-mers: peptide 92, peptide 93, peptide 94, peptide 95, peptide 96, peptide 97, peptide 98, 99 peptide and peptide 100; 10-mers: peptide 106, peptide 107, peptide 108, peptide 109, peptide 110, peptide 111, peptide 112, peptide 113 and peptide 114; 9-mers: peptide 122, peptide 123, peptide 124, peptide 125, 126 peptide, peptide 127, peptide 128 and peptide 129; 8-mers: peptide 139, 140 peptide, peptide 141, peptide 142, peptide 143, peptide 144 and peptide 145; 7-mers: peptide 157, peptide 158, peptide 159, peptide 160, peptide 161 and 162 peptide; 6-mers: peptide 176, peptide 177, peptide 178, peptide 179 peptide 180; 5-mers: peptide 196, peptide 197, peptide 198 and peptide 199; and 4-mers: peptide 217 peptide 219.

Bol is e preferred amino acid sequences of the selected peptides and chemically modified at the N - and/or-to the ends of the peptides according to the invention selected from the group consisting of 23-mers: peptide 2 and peptide 3; 22-mers: peptide 4 peptide 5 peptide 6; 21-mers: peptide 7 peptide 8, peptide 9 and peptide 10; 20-mers: peptide 11, peptide 12, peptide 13, peptide 14 and peptide 15; 19-mers: peptide 16, peptide 17, peptide 18, peptide 19, peptide 20 and peptide 21; 18-mers: peptide 22, peptide 23, peptide 24, peptide 25, peptide 26, peptide 27 and peptide 28; 17-mers: peptide 29, peptide 30, peptide 31, peptide 32, peptide 33, peptide 34, 35 peptide and peptide 36; 16-mers: peptide 37, peptide 38, peptide 39, 40 peptide: peptide 41, 42 peptide, peptide 43, 44 peptide and peptide 45; 15-mers: peptide 46, peptide 47, peptide 48, peptide 49, peptide 50, peptide 51, peptide 52, peptide 53 and peptide 54; 14-Mer: peptide 56, 57 peptide, peptide 58, peptide 59, peptide 60, peptide 61, 62 peptide, peptide 63 peptide 64; 13-mers: peptide 67, peptide 68, peptide 69, peptide 70, peptide 71, peptide 72, peptide 73, peptide 74, peptide 80, peptide 81, peptide 82, peptide 83, peptide 84, peptide 85, peptide 86 and peptide 87; 11-mers: peptide 92, peptide 93, peptide 94, peptide 95, peptide 96, peptide 97, peptide 98, 99 peptide and peptide 100; 10-mers: peptide 106, peptide 108, peptide 109, peptide 110, peptide 111, peptide 112, peptide 113 and peptide 114; 9-mers: peptide 124, peptide 125, 126 peptide, peptide 127, peptide 128 and peptide 129; 8-mers: peptide 141, peptide 142, peptide 143, peptide 144 and peptide 145; 7-mers: peptide 159, peptide 160, peptide 161 and 162 peptide; merov: peptide 178, peptide 179 peptide 180; 5-mers: peptide 198 and peptide 199; and 4-Mer: peptide 219.

In other embodiments of the invention, the amino acid sequence of the peptide comprises the contiguous residues of A,K,G and E of the reference sequence peptide 1 as in the peptide 219. For example, such peptides may have an amino acid sequence selected from the group consisting of: (a) amino acid sequences that have 4-23 contiguous amino acids of the reference sequence peptide 1, where the specified amino acid sequence of the peptide comprises the contiguous residues of A,K,G and E of the reference peptide 1 as in the peptide 219 (e.g., peptide 219, the peptide 45, the peptide 79, the peptide 67, the peptide 80 and the like); (b) a sequence essentially similar to the amino acid sequence defined in (a); and (c) options amino acid sequence defined in (a).

Examples of peptide segments containing the amino acid sequence AKGE reference peptide amino acid sequence, peptide 1, are (a): 23-measures: peptide 2 and peptide 3; 22-measures: peptide 4 peptide 5 peptide 6; 11-measures: peptide 7 peptide 8, peptide 9 and peptide 10; 20-measures: peptide 11, peptide 12, peptide 13, peptide 14 and peptide 15; 19-measures: peptide 16, peptide 17, peptide 18, peptide 19, peptide 20 and peptide 21; 18 - measures: peptide 22, peptide 23, peptide 24, peptide 25, peptide 26, peptide 27 and peptide 28; 17-measures: peptide 29, Atid 30, peptide 31, peptide 32, peptide 33, peptide 34, 35 peptide and peptide 36; 16-measures: 37 peptide, peptide 38, peptide 39, 40 peptide, peptide 41, 42 peptide, peptide 43, 44 peptide and peptide 45; 15-measures: peptide 46, peptide 47, peptide 48, peptide 49, peptide 50, peptide 51, peptide 52, peptide 53 and peptide 54; 14-measures: peptide 56, 57 peptide, peptide 58, peptide 59, peptide 60, peptide 61, peptide 62, 63 peptide and peptide 64; 13-measures: peptide 67, peptide 68, peptide 69, peptide 70, peptide 71, peptide 72, peptide 73, peptide 74 and peptide 75; 12-measures: peptide 79, peptide 80, peptide 81, peptide 82, peptide 83, peptide 84, peptide 85, peptide 86 and peptide 87; 11-measures: peptide 93, peptide 94, peptide 95, peptide 96, peptide 97, peptide 98, 99 peptide and peptide 100; 10-measures: peptide 108, peptide 109, peptide 110, peptide 111, peptide 112, peptide 113 and peptide 114; 9-measures: peptide 124, peptide 125, 126 peptide, peptide 127, peptide 128 and peptide 129; 8-measures: peptide 141, peptide 142, peptide 143, peptide 144 and peptide 145; 7-measures: peptide 159, peptide 160, peptide 161 and 162 peptide; 6-measures: peptide 178, peptide 179 and peptide 180; 5-steps: peptide 198 and peptide 199 and 4-Mer: peptide 219, (b) a sequence essentially similar to the amino acid sequence defined in (a); and (c) a variant amino acid sequence defined in (a)where the specified option is selected from the group consisting of variants with substitutions, variants with deletions, the option with the additions and combinations thereof, and where the specified segment contains 4-23 contiguous amino acids, or consists of these is minislot.

In another embodiment of the invention, a preferred peptide sequences have an amino acid sequence selected from the group consisting of (a) amino acid sequences that have 10-23 contiguous amino acids of the reference sequence peptide 1; (b) a sequence essentially similar to the amino acid sequence defined in (a)and (C) the variant amino acid sequence defined in (a)where the specified option is selected from the group consisting of variants with substitutions, variants with deletions and options with additions and combinations thereof, where the preferred amino acid sequences include the 23-Mer: peptide 2; 22-Mer: peptide 4; 21-Mer: peptide 7; 20-Mer: peptide 11; 19-Mer: peptide 16; 18-Mer: peptide 22; 17-Mer: peptide 29; 16-Mer: peptide 37; 15-Mer: peptide 46; 14-Mer: peptide 56; 13-Mer: peptide 67; 12-Mer: peptide 79; 11-Mer: peptide 92; and 10-Mer: peptide 106.

In other embodiments of the invention, the amino acid sequence of the peptide begins with the N-terminal amino acids of the reference sequence peptide 1 and includes adjacent residues A, K, G and E of the reference sequence peptide 1 as in the peptide 219, and in other embodiments of the invention, the amino acid sequence of the peptide ends at the C-terminal amino acids of the reference sequence peptide 1 and includes adjacent residues A, K, G and E atalo the Noah sequence of peptide 1 in the peptide 219.

These peptides may include one or more amino acid modifications, such as deletions, substitutions and/or additions introduced in the reference amino acid sequence. Preferred substitutions can be conservative amino acid substitutions, or such replacements can be non-conservative amino acid substitutions. In some embodiments of the invention, peptides, including peptides, amino acid sequence which is essentially identical to reference amino acid sequences, or their variants, have no deletions or additions, in contrast to the corresponding contiguous amino acids of the reference amino acid sequence, but may be conservative or not conservative replacement. Amino acid substitutions that can be entered in the reference amino acid sequence of the peptides according to the invention, include, but are not limited to, replacement of, where: alanine (A) can be replaced by a lysine (K), valine (V), leucine (L) or isoleucine (I); glutamic acid (E) can be replaced by aspartic acid (D); glycine (G) may be replaced by Proline (P); lysine (K) may be replaced by arginine (R), glutamine (Q) or asparagine (N); phenylalanine (F) may be replaced by a leucine (L), valine (V), isoleucine (I) or alanine (A); Proline (P) can be replaced by a glycine (G); glutamine (Q) can be replaced by glutamic acid (E) or asparagine (N); arginine (R) may be replaced by lysine (K), glutamine (Q) or asparagine (N); serine (S) may be replaced by threonine; threonine (T) can be replaced by a serine (S), and valine (V) may be replaced by a leucine (L), isoleucine (I), methionine (M), phenylalanine (F), alanine (A) or norleucine (Nle). For example, substitutions that can be entered in the reference amino acid sequence of the peptides according to the invention, are the replacement of the phenylalanine (F) by alanine (A) (for example, in position 4 amino acids of the reference amino acid sequence), the substitution of glutamine (Q), glutamic acid (E) (for example, at position 3 the amino acids of the reference amino acid sequence), the substitution of alanine (A), lysine (K) (for example, in positions 2 and/or 8 amino acids of the reference amino acid sequence), and/or substitution of threonine (T) by serine (S) (for example, in position 7 amino acids of the reference amino acid sequence).

If the amino acid sequences of the peptides according to the invention is administered replacement (where the peptides are unmodified and are peptides that have been chemically modified, for example, the introduction of N-terminal and/or C-terminal modification by formation of amide), preferably, when compared to the reference amino acid sequence identity IU the DN amino acid sequence of the indicated peptide and a reference amino acid sequence comprised of at least 80%. Peptides having 5-23 amino acids and comprising one amino acid substitution, about 80-96% (i.e. ~95,7%) identical to a reference amino acid sequence. Peptides having 10-23 amino acids and comprising one amino acid substitution, approximately 90-96% (i.e. ~95,7%) identical to a reference amino acid sequence. Peptides having 20-23 amino acids and comprising one amino acid substitution, about 95-96% (i.e. ~95,7%) identical to a reference amino acid sequence. Peptides having 10-23 amino acids and comprising two amino acid substitutions, about 80-92% (i.e. ~91,3%) identical to a reference amino acid sequence. Peptides having amino acids 16-23 and comprising two amino acid substitutions, approximately 87.5-92% (i.e. ~91,3%) identical to a reference amino acid sequence. Peptides having 20-23 amino acids and comprising two amino acid substitutions, about 90-92% (i.e. ~91,3%) identical to a reference amino acid sequence. Peptides having 15-23 amino acids and comprising three amino acid substitutions, about 80-87% identical to a reference amino acid sequence. Peptides having 20-23 amino acids and comprising three amino acid substitutions, approximately 85-87% identical to a reference amino acid sequence. Peptides having 20-23 amino acids and including couple the e amino acid substitutions, about 80-83% (i.e. ~82,6%) identical to a reference amino acid sequence.

The peptides according to the invention, namely in the amino acid sequence consisting of contiguous amino acids of the reference peptide (which is 24-Merom), replacement of one amino acid in the amino acid sequence consisting of contiguous 23 amino acids (23-Mer), selected from 24 amino acids of the reference amino acid sequence, results in a peptide amino acid sequence which 95,65% (or ~96%) identical to the amino acid segment of the reference peptide, which is identical to the specified 23-Mer. Similarly, the replacement of two, three, four and five amino acids in the specified 23-Mer yields a peptide with an amino acid sequence that is at 91,30% (or ~91%), 86,96% (or ~87%), 82,61% (or ~83%) and 78,27% (or ~78%) identical to, respectively, the amino acid sequence of the reference peptide. Similarly, the replacement of one, two, three, four and five amino acids in the specified 22-Mer yields a peptide with an amino acid sequence that is at 95,45% (~95%), 90,91% (or ~91%), 86,36% (or ~86%), 81,82% (or ~82%) and 77,27% (or ~77%)identical to, respectively, the amino acid sequence of the reference peptide. Similarly, the replacement of one, two, three, four and five amino acids specified in the om 21-Mer yields a peptide with the amino acid sequence, which 95,24% (~95%), 90,48% (~91%), 85,71% (~86%), 80,95% (~81%) and 76,19% (~76%) identical to, respectively, the amino acid sequence of the reference peptide. Similarly, the replacement of one, two, three, four and five amino acids in the specified 20-Mer yields a peptide with an amino acid sequence that is at 95,00% (95%), 90,00% (90%), 85,00% (85%), 80,00% (80%) and 75,00% (75%) identical to, respectively, the amino acid sequence of the reference peptide. Similarly, the replacement of one, two, three and four amino acids in the specified 19-Mer yields a peptide with an amino acid sequence that is at 94,74% (~95%), 89,47% (~89%), 84,21% (~84%) and 78,95% (~79%) identical to, respectively, the amino acid sequence of the reference peptide. Similarly, the replacement of one, two, three and four amino acids in the specified 18-Mer yields a peptide with an amino acid sequence that is at 94,44% (~94%), 88,89% (~89%), 83,33% (~83%) and 77,78% (~78%) identical to, respectively, the amino acid sequence of the reference peptide. Similarly, the replacement of one, two, three and four amino acids in the specified 17-Mer yields a peptide with an amino acid sequence that is at 94,12% (~94%), 88,23% (~88%), 82,35% (~82%) and 76,47% (~76%) identical to, respectively, the amino acid sequence of the reference peptide. Similarly, the replacement one is th, two, three and four amino acids at the specified 16-Mer yields a peptide with an amino acid sequence that is at 93,75% (~94%), 87,50% (~88%), 81,25% (~81%) and 75,00% (75%) identical to, respectively, the amino acid sequence of the reference peptide. Similarly, the replacement of one, two or three amino acids in the specified 15-Mer yields a peptide with an amino acid sequence that is at 93,33% (~93%), 86,67% (~87%) and 80.00% (80%) identical to, respectively, the amino acid sequence of the reference peptide. Similarly, the replacement of one, two or three amino acids in the specified 14-Mer yields a peptide with an amino acid sequence that is at 92,86% (~93%), 85,71% (~86%), and 78,57% (79%) identical to, respectively, the amino acid sequence of the reference peptide. Similarly, the replacement of one, two or three amino acids in the specified 13-Mer yields a peptide with an amino acid sequence that is at 92,31% (~92%), 84,62% (~85%), and 76,92% (~77%) identical to, respectively, the amino acid sequence of the reference peptide. Similarly, the replacement of one, two or three amino acids in the specified 12-Mer yields a peptide with an amino acid sequence that is at 91,67% (~92%), 83,33% (~83%) and 75,00% (75%) identical to, respectively, the amino acid sequence of the reference peptide. analogichnym way replacement of one or two amino acids at the specified 11-Mer yields a peptide with an amino acid sequence that is at 90,91% (~91%) and 81,82% (~82%) identical to, respectively, the amino acid sequence of the reference peptide. Similarly, replacement of one or two amino acids at the specified 10-Mer yields a peptide with the amino acid sequence, which 90,00% (90%) 80,00% (80%) identical to, respectively, the amino acid sequence of the reference peptide. Similarly, replacement of one or two amino acids at the specified 9-Mer yields a peptide with an amino acid sequence that is at 88, and 89% (~89%) and 77, 78% (~78%) identical to, respectively, the amino acid sequence of the reference peptide. Similarly, replacement of one or two amino acids at the specified 8-Mer yields a peptide with an amino acid sequence that is at 87,50% (~88%) and 75,00% (75%) identical to, respectively, the amino acid sequence of the reference peptide. Similarly, replacement of one amino acid at the specified 7-Mer, 6-Mer, 5-Mer and 4-Mer yields a peptide with an amino acid sequence that is at 85,71% (~86%), 83,33% (~83,3%), 80,00% (80%) and 75,00% (75%) identical to, respectively, the amino acid sequence of the reference peptide. Amino acid sequence according to the SNO invention, preferably, more than 80%, more preferably at 81-96%, and even more preferably, between 80 and 96% identical to reference amino acid sequence. Preferably, the amino acid sequence can be, but not necessarily, are chemically bonded at the N-terminal amino group of the peptide with linear C2-C22-aliphatic carboxylic acid group, more preferably linear With2-C16-aliphatic carboxylic acid group, and most preferably linear With2- or16-aliphatic carboxylic acid group through an amide bond, and optionally, chemically bonded at the C-terminal carboxylic acid group of the peptide with an amine, such as ammonia or with primary or secondary amine, such as1-C16-aliphatic primary amine with a straight chain through amide linkages.

Examples of variants of the peptide 79 with substitutions, namely 12-measure, are, for example, peptide 238, where the amino acid Q is present at position 3 of the peptide 79, in sequence 238 has been replaced with E; peptide 233, where the amino acid A, which is present in position 2 of the peptide 79, peptide 233 was replaced by K; peptide 234, where the amino acid A, which is present in position 8 of the peptide 79, peptide 234 has been replaced by K; peptide 235, where the amino acid A, which is present in positions 2 and 8 in the peptide 79, peptide 235 was replaced by K; peptide 237, where the amino acid F is present in position 4 of the peptide 79, peptide 237 was replaced with A; the peptide 239, where the amino acid K, which is present at the position 10 in peptide 79, peptide 239 has been replaced with A; the peptide 240, where the amino acid G, which is present at the position 11 in peptide 79, peptide 240 was replaced by A; and the peptide 241, where the amino acid E is present at the position 12 in peptide 79, peptide 241 was replaced by A.

Examples of variants of the peptide 106 with substitutions, namely 10-measure, are, for example, peptide 236, where the amino acid F is present in position 4 of the peptide 106, peptide 236 has been replaced with A; the peptide 242, where the amino acid G, which is present in position 1 of the peptide 106, peptide 237 was replaced with A; the peptide 243, where the amino acid Q is present at position 3 of the peptide 106, 243 peptide was replaced with A; the peptide 244, where the amino acid S, which is present in position 5 of the peptide 106 in the peptide 244 was replaced with A; the peptide 245, where the amino acid K, which is present at the position 6 in the peptide 106, peptide 245 has been replaced with A; the peptide 247, where the amino acid T, which is present in position 7 of the peptide 106, peptide 247 has been replaced with A; the peptide 248, where the amino acid K, which is present at the position 10 in the peptide 106, peptide 248 was replaced with A; the peptide 249, where the amino acid K, which is present in positions 6 and 10 in the peptide 106, peptide 249 has been replaced on A;

Note the ROM version of the peptide 137 with substitutions, namely 8-measure is, for example, peptide 250, where the amino acid F is present in position 4 of the peptide 137, 250 peptide was replaced by A.

An example of a variant peptide 219 with substitutions, namely 4-measure is, for example, peptide 251, where the amino acid K, which is present in position 2 of the peptide 219, peptide 251 has been replaced by A.

Variant peptides with substitutions, such as the variant described in this application can be obtained in the form of a selected peptide or in the form of chemically modified peptide, such as, for example, a peptide with N-terminal amidon, such as myristyl-amide, acetyl-amide, etc. as described in this application, and the peptide with C-terminal amidon, such as amide formed by ammonia, and the peptide with N-terminal and C-terminal amidon.

If the amino acid sequences of the peptides according to the invention include deletions, when compared to the reference amino acid sequence preferably, the identity between the amino acid sequence of the indicated peptide and a reference amino acid sequence comprised of at least 80%. Peptides having 5-23 amino acids and consisting of one amino acid deletions, about 80-96% (i.e. ~95,7%) identical to a reference amino acid sequence. Peptides having 10-23 amino acids and consisting of one amino acid deletions, when is Erno at 90-96% (i.e. ~95,7%) identical to a reference amino acid sequence. Peptides having 20-23 amino acids and consisting of one amino acid deletions, about 95-96% (i.e. ~95,7%) identical to a reference amino acid sequence. Peptides having 10-23 amino acids and comprising two amino acid deletions, about 80-92% (i.e. ~91,3%) identical to a reference amino acid sequence. Peptides having amino acids 16-23 and comprising two amino acid deletions, approximately 87.5-92% (i.e. ~91,3%) identical to a reference amino acid sequence. Peptides having 20-23 amino acids and comprising two amino acid deletions, approximately 90-92% (i.e. ~91,3%) identical to a reference amino acid sequence. Peptides having 15-23 amino acids and comprising three amino acid deletions, approximately 80-87% identical to a reference amino acid sequence. Peptides having 20-23 amino acids and comprising three amino acid deletions, approximately 85-87% identical to a reference amino acid sequence. Peptides having 20-23 amino acids and comprising four amino acid deletions, about 80-83% (i.e. ~82,6%) identical to a reference amino acid sequence.

As mentioned above, one or more amino acids of the peptides can be chemically modified. Amino acids peptides can be subjected to any modifications, a well-known specialist is m, using any known method.

In some embodiments of the invention, the N-terminal and/or C-terminal amino acid may be modified. For example, the alpha N-terminal amino acid peptides can be alkylated, liderovna or allerban the alpha N-terminal (N-terminal) amino(alpha-H2N-)groups and, for example, the C-terminal amino acids of these peptides can be liderovna or etherification the C-terminal carboxyl (-COOH) group. For example, the N-terminal amino group may be modified by acylation with amide formation, resulting in it will contain any acyl or fatty acyl group, including acetyl group (i.e. CH3-C(=O)- or meritorious group, which are the preferred groups from the point of view of the present invention). In some embodiments of the invention, the N-terminal amino group can be modified so that it will contain acyl group having the formula-C(O)R, where R is a straight or branched alkyl group having from 1 to 15 carbon atoms, or it can be modified so that it will contain acyl group having the formula-C(O)R1where R1represents a straight alkyl group having from 1 to 15 carbon atoms. N-amide can also be formamid (R=H). C-limit the I amino acid peptides can be chemically modified. For example, the C-terminal carboxyl group of the C-terminal amino acids can be chemically modified by conversion of the carboxyl group in carboxamido group (that is, it can be liderovna). In some embodiments of the invention, the N-terminal and/or C-terminal amino acids are not chemically modified. In some embodiments of the invention, the N-terminal group is modified and C-terminal group is not modified. In some embodiments of the invention are modified as N-terminal and C-terminal groups.

The peptide can be allerban the amino group of the N-terminal amino acids with the formation of the N-terminal amide by reaction with an acid selected from the group consisting of:

(i-a) C2(acetyl)-C13aliphatic (saturated or optionally unsaturated) carboxylic acids (for example, N-terminal amide formed by acetic acid (which is preferred), propanoic acid, butane acid, hexanoic acid, octanoic acid, decanoas acid, dodecanol acid), which may be straight or branched chain (more than3), or it may contain a ring (more than3);

(i-b) rich C14aliphatic carboxylic acids, which may have direct or rasvet the feudal chain, or it may contain a ring;

(i-c) unsaturated C14aliphatic carboxylic acids which may be straight or branched chain, or it may contain a ring;

(i-d) C15-C24aliphatic (saturated or optionally unsaturated) carboxylic acid, which may be straight or branched chain, or it may contain a ring (for example, tetradecanoic acid (myristic acid, which is the preferred group), with hexadecanoic acid, 9-hexadecenoic acid, with octadecanoic acid, 9-octadecenoic acid, 11-hexadecenoic acid, 9,12 - octadecadienoic acid, 9,12,15-octadecatrienoic acid, 6,9,12 - octadecatrienoic acid, with eicosanol acid, 9-Aksenovo acid, 5,8,11,14-eicosatetraenoic acid, 5,8,11,14,17-eicosapentaenoic acid, with docosanoic acid, 13-docosenoic acid, 4,7,10,13,16,19-docosahexaenoic acid, with tetracosanoic acid and the like);

(ii) triperoxonane acids;

(iii) benzoic acid; and

(iv-a) Cl-C12aliphatic alkylsulfonic acid, which forms an aliphatic alkylsulfonate where the structure of the Cl-C12aliphatic alkyl chain sulfonic acid is similar to the structure of the aliphatic alkyl chain carboxylic acid described above and efficiency alkylcarboxylic acids. For example, the peptide can be allerban using a carboxylic acid group represented by the formula (C1-C11)alkyl-C(O)OH, by the dehydration reaction by activating the carboxylic acid group with the formation of amide represented by the formula (C1-C11)alkyl-C(O)-NH-peptide. Similarly, the sulfonamide can be obtained through the interaction of groups of sulfonic acid represented by formula (C1-C12)alkyl-S(O2)-X, for example, where X represents a halogen or OCH3or other compatible leaving group) with N-terminal amino group with the formation of sulfonamida represented by the formula (C1-C12)alkyl-S(O2)-NH-peptide;

(iv-b) Cl4-C24aliphatic alkylsulfonic acid, which forms an aliphatic alkylsulfonate where the structure of the Cl4-C24aliphatic alkyl chain sulfonic acid is similar to the structure of the aliphatic alkyl chain carboxylic acid in the above-described aliphatic alkylcarboxylic acids. For example, the peptide can be allerban using a carboxylic acid group represented by the formula (C13-C23)alkyl-C(O)OH, by the dehydration reaction by activating the carboxylic acid group with the formation of amide represented by the formula (C13 -C23)alkyl-C(O)-NH-peptide. Similarly, the sulfonamide can be obtained through the interaction of groups of sulfonic acid represented by formula (C14-C24)alkyl-S(O2)-X, for example, where X represents a halogen or OCH3or other compatible leaving group) with N-terminal amino group with the formation of sulfonamida represented by the formula (C14-C24)alkyl-S(O2)-NH-peptide.

In another example, the N-terminal amino group of the N-terminal amino acids can be alkylated With1-C12aliphatic alkyl group, the structure of which is described above. The alkylation can be carried out, for example, aliphatic alkylhalogenide or aliphatic Olkiluoto ether sulfonic acid (nelfinavir, tosilata and the like), and preferably, using primary alkylhalogenide or primary Olkiluoto ether sulfonic acid. N-terminal amino acid may be modified at the terminal amino group so that it contains as amide any acyl or aliphatic acyl or fatty acyl group, including acetyl group (i.e.- C(O)CH3that is the preferred group), meritorious group (which is the preferred group), butanoyloxy group, hexanoyl group, octane is inuu group, technology group, dodecanoyl group, tetradecanoyl group, hexadecanoyl group, 9-hexadecanoyl group, octadecanoyl group, 9-octadecanoyloxy group, 11-octadecanoyloxy group, 9,12-octadecadienyl group, 9,12,15-octadecatrienoic group, 6,9,12-octadecatrienoic group, eicosanol group, 9-eicosanol group, 5,8,11,14-eicosatetraenoic group, 5,8,11,14,17-eicosapentaenoic group, docosanol group, 13-docosenoic group, 4,7,10,13,16,19-docosahexaenoyl group and tetracosanoic group, where these groups are covalently bound to the terminal amino group of a peptide amide bond.

C-terminal group of the carboxylic acid C-terminal amino acids of the peptides according to the invention can also be chemically modified. For example, the C-terminal amino acid may be chemically modified through interaction of C-terminal carboxylic acid group of the peptide with the amine with the formation of amide groups, such as amide ammonia, which is a preferred group; amide With1-C12aliphatic alkylamine, preferably, aliphatic alkylamine with a straight chain; amide hydroxyl-substituted C1-C12aliphatic alkylamine; amide 2-C1-C12aliphatic alkylenediamine with direct chain and the amide omegamatic-poly(ethylenoxy) n-etilenovomu (also called omega-methoxy-PEG-alpha-amino group or an omega-methoxy-(polyethylene glycol)to the amino group), where n is 0-10. C-terminal carboxylic acid group of the C-terminal amino acids of the peptide may also be present in the form of ester selected from the group consisting of ether complex1-C12aliphatic Olkiluoto alcohol and ether complex of 2-(omega-methoxy-poly(ethyleneoxy)n-ethanol (MPEG), where n is 0-10. In one aspect of the invention, polietilenglikolya component, such as component, present in a complex ester of PEG, in a complex ester MPEG, amide PEG, amide MPEG and the like, preferably has a molecular weight of from about 500 to 40,000 daltons, more preferably from 1000 to 25000 daltons, and most preferably from about 1000 to 10,000 daltons.

C-terminal carboxylic acid group on the peptide, which may be represented by the formula peptide-C(O)OH, may be liderovna by means of its transformation into an activated form such as a carboxylic acid halide, carboxylic acid anhydride, N-hydroxysuccinimidyl, pentafluorophenyl (OPfp) ester, ester with 3-hydroxy-2,3-dihydro-4-oxo-benzotriazole (ODhbt) and the like, to facilitate interaction with ammonia or with primary or secondary amine, preferably ammonia or a primary amine, if e is ω, preferably, any other reactive group of the peptide were protected chemically synthesized compatible protective groups are well known to experts in the field of peptide synthesis, and in particular solid-phase peptide synthesis, and such groups are benzyl ether, tert-butyl methyl ether, phenyl ether and the like, Amide derived peptide may be represented by formula peptide-C(O)-NR3R4(amide is present at the C-end of the peptide), where R3and R4independently selected from the group consisting of hydrogen, C1-C12of alkyl, such as methyl, ethyl, butyl, isobutyl, cyclopropylmethyl, hexyl, dodecyl and, optionally, higher alkyl, for example With14-C24of alkyl, such as tetradecyl and the like described above.

The C-terminal carboxylic acid C-terminal amino acids can also be converted into amide hydroxyl-substituted C2-C12aliphatic alkylamine (where the hydroxyl group attached to the carbon atom, not the nitrogen atom of amine), such as 2-hydroxyethylamine, 4-hydroxyethylamine and 12-hydroxycobalamin etc.

The C-terminal carboxylic acid can also be converted into amide hydroxy-substituted C2-C12aliphatic alkylamine, where the hydroxyl group may be allerban with the formation of ester C2-C 12aliphatic carboxylic acids described above. Preferably, in amide peptide at the C-end of the indicated peptide represented by the formula peptide-C(O)NR5R6, R5represents hydrogen, and R6selected from the group consisting of hydrogen, C1-C12of alkyl and hydroxyl-substituted C2-C12the alkyl.

The C-terminal carboxylic acid C-terminal amino acids can also be converted into amide 2-(C2-C12)aliphatic alkylenediamine with a straight chain. This amide can be obtained, for example, by interacting linear C2-C12aliphatic alcohol with potassium hydride in diglyme with 2 chloroethanol, with the formation of C2-C12aliphatic alkylamine with direct chain, which can be converted into amine by oxidation of the aldehyde followed by reductive amination to an amine (for example, using ammonia) or by transformation in alkylhalogenide (for example, using thionyl chloride) followed by treatment of an amine, such as ammonia.

The C-terminal carboxylic acid C-terminal amino acids can also be converted into amide linear PEG-amine (for example, omega-hydroxy-PEG-alpha-amine, omega-(C2-C12)-PEG-alpha-amine, such as omega-methoxy-PEG-alpha-amine, i.e. MPEG-Amin). In one aspect of the invention, polietilenglikolya component or PEG preferably have a molecular weight from about 500 to 40,000 daltons, more preferably from 1000 to 25000 daltons, and most preferably from about 1000 to 10,000 daltons.

The C-terminal carboxylic acid C-terminal amino acids can also be converted into amide omega-methoxy-poly(ethyleneoxy)n-ethylamine, where n is 0-10, which can be obtained from the corresponding omega-methoxy-poly(ethyleneoxy)nethanol, for example, by conversion of the alcohol to the amine as described above.

In another embodiment of the invention, the C-terminal carboxyl can be transformed into an amide represented by the formula peptide-C(O)-NR7R8where R7represents hydrogen, and R8is a linear 2-(C1-C12)aliphatic alkyloxyalkyl group where the specified C1-C12aliphatic alkyl group defined above, and includes such groups as methoxyethyl (i.e. CH3O-CH2CH2), 2-dodecyloxy and the like, or R7represents hydrogen, and R8is an omega-methoxy-poly(ethyleneoxy)n-ethyl group, where n poly(ethyleneoxy)groups is 0-10, and where such groups are 2-methoxyethyl (i.e. CH3O-CH2CH2), omega-methoxyethoxymethyl (i.e. CH3O-CH2CH2O-CH2 CH2- CH3O-(CH2CH2O)10-CH2CH2-).

C-terminal carboxylic acid group of the C-terminal amino acids of the peptide may also be present in the form of ester C1-C12aliphatic Olkiluoto alcohol where the specified aliphatic alkyl group of the alcohol defined above. C-terminal carboxylic acid group of the C-terminal amino acids of the peptide may also be present in the form of ester 2-(omega-methoxy-poly(ethyleneoxy)n)ethanol, where n is 0-10, which can be obtained by reaction of 2-methoxyethanol, such as 2-methoxyethanol sodium, with stoichiometric quantities of ethylene oxide, where the aforementioned stoichiometric amount depends on the number n.

Side chain of amino acids, peptides may be chemically modified. So, for example, phenyl group of phenylalanine or tyrosine may be substituted by the Deputy selected from the group consisting of:

C1-C24aliphatic alkyl groups (that is, a straight or branched group, and/or saturated or unsaturated groups and/or groups containing a cyclic group, such as methyl (preferred), ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, 2-methylcyclopropyl, cyclohexyl, octyl, decyl, dodecyl, hexadecyl, octadecyl, eicosanol, docosanol, t is trazonil, 9-hexadecanoyl, 9-octadecenoyl, 11-octadecenoyl, 9,12-octadecadienyl, 9,12,15-octadecatrienoic, 6,9,12-octadecatrienoic, 9-eicosene, 5,8,11,14-eicosatetraenoic, 5,8,11,14,17-eicosapentaenic, 13-docosanol and 4,7,10,13,16,19 - docosahexaenic;

C1-C12aliphatic alkyl groups, substituted hydroxyl group at least one carbon atom located at a certain distance from the unsaturated position, and examples of such hydroxyalkyl groups are hydroxymethyl, hydroxyethyl, hydroxydiphenyl etc.;

C1-C12alkyl groups, substituted hydroxyl group which is esterified C2-C25aliphatic carboxyl group of the acid, such as acetic acid, butane acid, hexanoic acid, octanoic acid, cekanova acid, dodekanisa acid, tetradecanoic acid, hexadecanol acid, 9-hexadecanoate acid, octadecanoic acid, 9-octadecenoate acid, 11-octadecenoyl acid, 9,12-octadecadienoic acid, 9,12,15-octadecatrienoic acid, 6,9,12-octadecatrienoic acid, Aksenova acid, 9-Aksenova acid, 5,8,11,14-eicosatetraenoic acid, 5,8,11,14,17-eicosapentaenoic acid, docosanoate acid, 13-docosanoate acid, 4,7,10,13,16,19-docosahexaenoic acid, tetracosanoic acid and the like, dicarboxylic acid, such a spacecraft is succinic acid, or hydroxy acids such as lactic acid, where the total number of carbon atoms of the ester substituent is 3-25;

halogen, such as fluorine, chlorine, bromine and iodine;

nitro;

amino, such as NH2methylamino, dimethylamino;

trifloromethyl;

carboxyl (-COOH);

With1-C24alkoxy (for example, alkoxy, which can be formed by alkylation of tyrosine), such as methoxy, ethoxy, propyloxy, isopropoxy, bucalossi, isobutoxy, cyclopropylamino, 2-methoxycyclohexyl, cyclohexyloxy, octyloxy, decyloxy, dodecyloxy, hexadecylamine, octadecylamine, eicosanoic, docosanoic, tetracosanoic, 9 hexadecenoic, 9 octadecanoyloxy, 11 octadecanoyloxy, 9,12-octadecadienoate, 9,12,15-octadecatrienoic, 6,9,12-octadecatrienoic, 9 eicosenoic, 5,8,11,14-eicosatetraenoate, 5,8,11,14,17-eicosapentaenoate, 13 docosenoic and 4,7,10,13,16,19-docosahexaenoate; and

C2-C12hydroxyalkyloxy, such as 2-hydroxyethyloxy and esters of carboxylic acids described above, or esters triperoxonane acid.

The hydroxyl group of serine can be etherification Deputy selected from the group consisting of:

C2-C12aliphatic carboxylic acid group described above;

group triperoxonane acid; and

group is s benzoic acid.

The Epsilon-amino group of lysine can be chemically modified, for example, through the formation of amide by reaction with C2-C12aliphatic carboxylic acid group (for example, through the interaction of the amine with a chemically activated form of a carboxylic acid such as the acid chloride, the anhydride, N-hydroxysuccinimidyl, pentafluorophenyl (OPfp) ester, ester with 3-hydroxy-2,3-dihydro-4-oxo-benzotriazole (ODhbt) and the like), for example, described above, or with a group of benzoic acid or amino acid group. In addition, the Epsilon-amino group of lysine can be chemically modified by alkylation of one or two1-C4aliphatic alkyl groups.

The group of carboxylic acid to glutamic acid can be modified through education amide by reacting with an amine, such as ammonia, primary C1-C12aliphatic alkylamine (the alkyl group defined above), including methylamine, or with the amino group of amino acids.

The group of carboxylic acid to glutamic acid can be modified by formation of ester by interacting with C2-C12aliphatic hydroxyalkyl group described above, and preferably, with the primary C1-C12aliphatic Akilov the m alcohol, such as methanol, ethanol, propan-1-ol, n-dodecanol, and the like described above.

In its preferred embodiment, the present invention relates to a method of inhibiting the release of at least one mediator of inflammation of granules of at least one inflammatory cells in the tissue and/or body fluids of an individual, where the method includes an introduction to the specified fabric and/or a physiological fluid therapeutically effective amount of a pharmaceutical composition containing at least one peptide having an amino acid sequence selected from the group consisting of:

(a) amino acid sequences that have 4-23 contiguous amino acids of the reference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1);

(b) amino acid sequences that have sequence GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1); and

(c) amino acid sequence essentially identical to the sequence defined in (a), where C-terminal amino acid of the peptide is, but not necessarily, independently chemically modified and the N-terminal amino acid of the peptide or is independently chemically modified by acylation with carboxylic acid selected from the group consisting of C2-C13saturated or unsaturated aliphatic carboxylic acid, With14 saturated (myristic acid) or unsaturated aliphatic carboxylic acids, C15-C24saturated or unsaturated aliphatic carboxylic acid, and triperoxonane acid, or is not chemically modified; provided that the peptide can be modified by acylation, if its amino acid sequence begins with a sequence GAQF reference sequence, i.e. by only acylation of carboxylic acid selected from the group consisting of C2-C13saturated or unsaturated aliphatic carboxylic acid, With14unsaturated aliphatic carboxylic acid, C15-C24saturated or unsaturated aliphatic carboxylic acids and triperoxonane acid, or acid, which is not chemically modified, where the specified peptide United, but not necessarily, with a pharmaceutically acceptable carrier and is present in an amount that is therapeutically effective to attenuate release of inflammatory mediators and thereby to reduce the release rate of the specified mediator of inflammation of at least one inflammatory cells compared with the release rate of the specified mediator of inflammation of at least one inflammatory cells of the same ti is a, but in the absence of at least one peptide.

In this way, preferably, is used in the peptide, which can be acetiminophen the alpha N-terminal amino acids. This peptide may consist of at least ten contiguous amino acid residues, and preferably represents acetyl-peptide 106 (SEQ ID NO:106).

This method is also used a peptide consisting of at least four contiguous amino acid residues, and more preferably, of at least six contiguous amino acid residues. In addition, this peptide can be meritorious the alpha N-terminal amino acids of the indicated peptide. This method can also be used peptide, which can be amitirova ammonia in the alpha C-terminal amino acids.

In the method according to another variant of the invention is a peptide which contains an amino acid sequence selected from (a) amino acid sequences that have 4-23 contiguous amino acids of the reference sequence GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1), where N-terminal amino acid of amino acid sequence (a) is selected from the amino acids present at positions 2-21 reference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1). In addition, these peptides can be meritorious the alpha N-terminal amino acids and can also be lidirovali what miacom the alpha C-terminal amino acids.

The method of administration according to the invention is aimed at reducing the release of mediators of inflammation by blocking mechanism or inhibition of release of mediators of inflammation inflammatory cells in the specified individual.

This method includes the introduction in the described peptides pharmaceutically acceptable carrier or mix of these peptides with the specified media with obtaining pharmaceutical compositions.

The method of administration according to the invention is aimed at reducing the release of at least one mediator of inflammation and thereby to reduce the release rate of the specified mediator of inflammation of at least one inflammatory cells compared with the release rate of the specified mediator of inflammation of at least one inflammatory cells of the same type, but in the absence of at least one of the indicated peptide. Inflammatory cell specific individual can be the leukocyte, granulocyte, basofil, eosinophil, monocyte, macrophage, or combinations thereof.

Mediator of inflammation released from at least one of the granules of at least one inflammatory cells selected from the group consisting of myeloperoxidase (MPO), peroxidase eosinophils (EPO), the main major protein [MBP], lysozyme, granzyme, histamine, proteoglycan, protease, he is taxisco factor, cytokine, a metabolite of arachidonic acid, defensin, protein, increasing permeability of the bacteria (BPI), elastase, cathepsin G, cathepsin B, cathepsin D, beta-D - glucuronidase, alpha-mannosidase, phospholipase A2, chondroitin-4-sulfate, proteinase 3, lactoferrin, collagenase, activator of complement, complement receptor, receptor N-formylmethyl-leucyl-phenylalanine (FMLP), laminin receptor, cytochrome b558, macrophage-chemotactic factor, histaminase, protein, bind with vitamin B12, gelatinase, plasminogen activator, beta-D-glucuronidase, and combinations thereof. The preferred mediator of inflammation is selected from the group consisting of myeloperoxidase (MPO), peroxidase eosinophils (EPO), the main major protein (MBP), lysozyme, granzyme and their combinations.

The method according to item 13 claims, where the effective amount of the indicated peptide, reduces the level of release of mediators of inflammation, includes inhibiting the degranulation of the amount of peptide that reduces the level of a mediator of inflammation released from at least one inflammatory cells, about 1%-99%, or preferably about 5-50% - 99%, compared to the amount released at least one inflammatory cell in the absence of the indicated peptide.

The method according to the invention can be applied in the t to be applied for the treatment of the individual, suffering from respiratory illness. This respiratory disease may be asthma, chronic bronchitis, chronic obstructive pulmonary disease (COPD) and cystic fibrosis. Preferred by individuals who may be subjected to treatment in accordance with the present invention are mammals, like humans, dogs, horses and cats.

By way of introduction of the peptides according to the invention is a local injection, parenteral administration, rectal administration, intra-lungs introduction, intranasal introduction and oral administration. More preferably, intra-lungs introduction includes the introduction of spray that can be carried out using insufflator, nebulizer with dosing valve or a nebulizer. In addition, the introduction of the individual can also include the introduction of a second molecule selected from the group consisting of antibiotics, antiviral compounds, antiparasitic compounds, anti-inflammatory compounds and immunomodulator.

This method can also be applied to treatment of an individual suffering from a disease selected from the group consisting of intestinal diseases, skin diseases, autoimmune diseases, pain syndrome, and combinations thereof. More specifically, the specified intestinal disease selected from the gr is PPI, consisting of ulcerative colitis, Crohn's disease and irritable bowel syndrome. Skin diseases, which can also be treated by the method according to the invention are rosacea, eczema, psoriasis and acne severe. In addition, the individual suffering from rheumatoid arthritis, may also be subjected to treatment by the method according to the invention.

In one of its variants, the present invention includes the introduction of peptides containing the amino acid sequence essentially identical to the amino acid sequence: (a) having 4-23 contiguous amino acids of the reference sequence GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1). These peptides, preferably selected from the group consisting of SEQ ID NOS:233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 247, 248, 249, 250, 251 and 252. These peptides can also be azetilirovanny for alpha N-terminal amino acid or meritorious for alpha N-terminal amino acid and optional lidirovali ammonia on alpha C-terminal amino acid.

The method according to the invention can also be applied to reduce the hypersecretion of mucus in the individual by introducing described herein peptides according to the invention, and also to reduce the level of MARCKS-associated hypersecretion of mucus at least one of the mucus-secreting cells or tissue of the individual, so that the hypersecretion of mucus in the specified individual was decreased compared with mucus hypersecretion observed in the absence of the indicated peptide.

The present invention relates to selected peptide having the amino acid sequence selected from the group consisting of:

(a) amino acid sequences that have 4-23 contiguous amino acids of the reference sequence GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1);

(b) amino acid sequences that have sequence GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1); and

(c) amino acid sequence essentially identical to the sequence defined in (a),

where C-terminal amino acid of the peptide is, but not necessarily, independently chemically modified and the N-terminal amino acid of the peptide or is independently chemically modified by acylation with carboxylic acid selected from the group consisting of C2-C13saturated or unsaturated aliphatic carboxylic acid, With14saturated (myristic acid) or unsaturated aliphatic carboxylic acids, C15-C24saturated or unsaturated aliphatic carboxylic acid, and triperoxonane acid, or is not chemically modified, provided that the peptide is modified by acylation, if its amino acid sequence begins with a sequence GAQF reference sequence, that is here by acylation only carboxylic acid, selected from the group consisting of C2-C13saturated or unsaturated aliphatic carboxylic acid, With14unsaturated aliphatic carboxylic acid, C15-C24saturated or unsaturated aliphatic carboxylic acids and triperoxonane acid, or acid, which is not chemically modified, where the specified peptide optionally combined with a pharmaceutically acceptable carrier and is present in an amount that is therapeutically effective to attenuate release of inflammatory mediators and thereby to attenuate release rate specified mediator of inflammation of at least one inflammatory cells compared with the release rate of the specified mediator of inflammation of at least one inflammatory cells of the same type, but in the absence of at least one specific peptide.

The selected peptide can be acetiminophen the alpha N-terminal amino acids. This highlighted the peptide consists of at least ten contiguous amino acid residues, and preferably represents a selected peptide, which consists of acetyl-peptide 106 (SEQ ID NO:106).

In another embodiment of the invention, the said peptide consists of at least four contiguous amino acid residues, or at least of six Smin the x amino acid residues.

Such a peptide can also be meritorious the alpha N-terminal amino acids and/or it can be amitirova ammonia in the alpha C-terminal amino acids.

The selected peptide may also contain amino acid sequence described in (a) and having 4-23 contiguous amino acids of the reference sequence GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1), where N-terminal amino acid of amino acid sequence (a) is selected from the amino acids present at positions 2-21 reference sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1). This peptide can also be meritorious or acetiminophen the alpha N-terminal amino acids or, but not necessarily, amitirova ammonia in the alpha C-terminal amino acids.

In another embodiment, the present invention relates to selected peptide, amino acid sequence which is essentially identical to the amino acid sequence (a)having 4-23 contiguous amino acids of the reference sequence GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1). These peptides, preferably selected from the group consisting of SEQ ID NOS:233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 247, 248, 249, 250, 251 and 252. These peptides can also be azetilirovanny the alpha N-terminal amino acids or meritorious the alpha N-terminal amino acids and optional lidirovali ammonia in the alpha C-terminal amino acids. Amino acid pic is egovernance (C), essentially identical to the amino acid sequence of (a)selected from the group consisting of SEQ ID NOS: 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 247, 248, 249, 250, 251 and 252.

The present invention also relates to compositions comprising the selected peptide described in the present application, and filler. The present invention also relates to pharmaceutical compositions containing the selected peptide described in the present application, and a pharmaceutically acceptable carrier. The pharmaceutical composition may also be preferably sterile, sterilized or sterilized. These peptides can be included in the set together with reagents suitable for injection.

Brief description of the graphical material

On figa-1B illustrated that PKC-dependent phosphorylation leads to the release of MARCKS from the plasma membrane into the cytoplasm.

On figa-2C shows that PKG induces the dephosphorylation of MARCKS under the action of activating PP2A.

Figure 3 presents histograms, which illustrated that PP2A is a major component of the path secretion of mucin.

Figure 4 shows the gel, which illustrates that MARCKS is associated with actin and myosin in the cytoplasm.

Figure 5 illustrates the mechanism of signal transmission, regulating the secretion of MPO in neutrophils.

Figure 6 presents histogra the mA to illustrate the ability of the MANS peptide to block the secretion of myeloperoxidase from selected canine neutrophils.

Figure 7 presents a histogram illustrating the ability of the MANS peptide to block the secretion of myeloperoxidase from selected human neutrophils.

On Fig presents the histogram, indicating that PMA stimulates a small increase in the level of MPO secretion from LPS-stimulated human neutrophils, which is enhanced by co-stimulation under the action of 8-Br-cGMP, depending on concentration.

Figure 9 presents the histogram, indicating that 8-Br-cGMP stimulation has little effect on the MPO secretion from LPS-stimulated human neutrophils until then, until you reached costimulate under the action of PMA depending on concentration.

Figure 10 presents the histogram, indicating that PMA stimulates a small increase in the level of MPO secretion from LPS-stimulated canine neutrophils, which increases costimulate under the action of 8-Br-cGMP, depending on concentration.

Figure 11 presents the histogram, indicating that 8-Br-cGMP stimulation has little effect on the MPO secretion from LPS-stimulated canine neutrophils until then, until you reached costimulate under the action of PMA depending on to whom centration.

On Fig presents the histogram, indicating that costimulate under the action of PMA+8-Br-cGMP is required for maximum MPO secretion from LPS-stimulated canine neutrophils.

Detailed description of the invention

Below is a more detailed description of the present invention with reference to the graphical material, in which is illustrated the preferred embodiments of the invention. However, the present invention can be implemented in different ways, which should not be construed as limiting the scope of the invention. On the contrary, these options are given for more detailed and complete description of the invention and in its entirety are included in the scope of the present invention.

If it is not specifically mentioned, all used here is the technical and scientific terms have their conventional meanings that are understandable to the average expert in the field that applies the present invention. All publications, patent applications, patents and other mentioned here work in its entirety introduced into the present description by reference.

The present invention relates to a method of inhibiting ekzoticheskogo release of at least one mediator of inflammation of at least one inflammatory cells, where the method includes contacting for men is our least one inflammatory cells, containing at least one mediator of inflammation that is present in the intracellular vesicles, at least one peptide selected from the group consisting of MANS peptide and its active fragment in amounts effective to reduce the release of mediators of inflammation inflammatory cells, compared with the level of release of mediators of inflammation inflammatory cells of the same type observed in the absence of at least one peptide.

The present invention also relates to a method of inhibiting the release of at least one mediator of inflammation of at least one inflammatory cells in the tissue or body fluids of an individual, where the method includes introducing into the tissue and/or a physiological fluid of an individual, with at least one inflammatory cell comprising at least one mediator of inflammation that is present in the intracellular vesicles, a therapeutically effective amount of a pharmaceutical composition containing at least one peptide selected from the group consisting of MANS peptide and its active fragment in the amount therapeutically effective to reduce the release of mediators of inflammation by at least one inflammatory cells, compared with the release rate honey is atora inflammation of at least one inflammatory cells of the same type in the absence of at least one peptide. More specifically, reducing the release of mediators of inflammation involves blocking or inhibition mechanism of release of mediators of inflammation from inflammatory cells.

The present invention relates to communication described in this application of the peptide with any known inflammatory cell and/or to the introduction of this peptide in the specified cell, which may be present in tissue or body fluids of an individual, with at least one mediator of inflammation that is present in the intracellular vesicles. Such inflammatory cell, preferably, is a leukocyte, and more preferably, granulocyte, which can also be classified as a neutrophil, basophil, eosinophil or combinations thereof. Inflammatory cells exposed to contact by the method according to the invention can be also monocytes/macrophages.

The present invention relates to the reduction in release of inflammatory mediators contained in the vesicles of inflammatory cells and inflammatory mediators selected from the group consisting of myeloperoxidase (MPO), peroxidase eosinophils (EPO), the main major protein (MBP), lysozyme, granzyme, histamine, proteoglycan, proteases, chemotactic factor, a cytokine, a metabolite of arachidonic acid, defensin, protein, increasing the surrounding permeability of bacteria (BPI), elastase, cathepsin G, cathepsin B, cathepsin D, beta-D-glucuronidase, alpha-mannosidase, phospholipase A2, chondroitin-4-sulfate, proteinase 3, lactoferrin, collagenase, activator of compellent, receptor complement receptor N-formylmethyl-leucyl-phenylalanine (FMLP), laminin receptor, cytochrome b558, macrophage-chemotactic factor, histaminase, protein, bind with vitamin B12, gelatinase, plasminogen activator, beta-D-glucuronidase, and combinations thereof. Preferred inflammatory mediators selected from the group consisting of myeloperoxidase (MPO), peroxidase eosinophils (EPO), the main major protein (MBP), lysozyme, granzyme and their combinations.

The present invention relates to communication effective amount of the peptide with inflammatory cell, where the specified effective amount is defined as inhibiting the degranulation number of MANS peptide or an active fragment, which reduces the level of a mediator of inflammation released from at least one inflammatory cells about 1%-99% compared to the level of a mediator of inflammation released from at least one inflammatory cell in the absence of MANS peptide or an active fragment. This number is also known as amount effective to reduce the release of the mediator of the inflammation. More preferably, the effective amount of the contact of the peptide includes inhibiting the degranulation number of MANS peptide or an active fragment, which reduces the level of a mediator of inflammation released from at least one inflammatory cells by about 5-50% - 99%, compared to the level of a mediator of inflammation released from at least one inflammatory cell in the absence of MANS peptide or an active fragment.

In one of its variants, the present invention relates to a method of introducing at least one peptide containing the MANS peptide and its active fragment in therapeutically effective amount, in fabric or in a physiological fluid of an individual suffering from a respiratory disease, which, preferably, is asthma, chronic bronchitis or COPD. In another embodiment of the invention, the individual may suffer intestinal disease, skin disease, autoimmune disease, pain, and combinations thereof. Specified intestinal disease may be ulcerative colitis, Crohn's disease or irritable bowel syndrome. The individual may suffer skin disease such as rosacea, eczema, psoriasis or acne severe. The individual may also suffer from arthritis, such as rheumatoid arthritis, psoriatic the ski arthritis and systemic lupus erythematosus. Individuals suffering from cystic fibrosis, may also be treated by the method according to the invention and the treatment with peptides. The method according to the invention, preferably, can be applied for the treatment of individuals, such as mammals, and preferably, people, dogs, horses and cats.

In accordance with the present invention, a method for the treatment of individuals is the introduction of one or more of the peptides described here including the MANS peptide or an active fragment, and such techniques are local injection, parenteral administration, rectal administration, intra-lungs introduction, intranasal administration or oral administration. More preferably, intra-lungs introduction selected from the group consisting of injection and spray, insufflator, nebulizer with dosing valve and the nebulizer. In addition, the described method may also include the introduction of a specified individual, the second molecule is selected from the group consisting of antibiotics, antiviral compounds, antiparasitic compounds, anti-inflammatory compounds and immunomodulator.

In one of its aspects, the present invention relates to a method of administration of the pharmaceutical composition. Such a pharmaceutical composition includes a therapeutically effective the top the number of known compounds and a pharmaceutically acceptable carrier. Used herein, the term "therapeutically effective" amount refers to the amount of the compound that is sufficient to alleviate the symptoms observed in the individual. Such therapeutically effective amount may vary depending on age and physical condition of the patient, and the severity of the condition of the patient being treated, the duration of the treatment of any type at the same time of treatment, used pharmaceutically acceptable carrier and other factors, known or established expert in this field. Pharmaceutically acceptable preparations, preferably, are of solid dosage forms such as tablets or capsules. For oral administration can also be used in liquid preparations, which may be received in the form of syrups or suspensions, for example solutions containing the active ingredient, sugar and a mixture of ethanol, water, glycerol and propylene glycol. If necessary, such liquid preparations may include one or more of the following agents, such as dyes, fragrances and saccharin. In addition, can also be used thickeners, such as carboxymethylcellulose, and other appropriate media, the choice of which can be carried out by a specialist in D. the authorized area.

As mentioned above, the present invention relates to a method of regulating cellular secretory processes, in particular processes for the release of inflammatory mediators from inflammatory cells. Used herein, the term "regulation" means blocking, inhibiting, reducing, attenuation, increase, increased, or stimulation. A number of cellular secretory processes includes the release of the content of membrane-bound vesicles or granules in the cells. Membrane-bound vesicles or granules defined as intracellular particle, which is mainly vesicular (or vesicles present in the cell) and contains extra material that can be secreted. It was found that the specific content of these vesicles, for example, present in inflammatory cells, is responsible for the development of various pathologies in several mammalian tissue. Some effects such secretion, obviously, are lesions previously normal tissue during inflammation. The present invention relates to a method of blocking the secretion of any membrane-bound vesicles, including vesicles present in inflammatory cells by targeting synthetic peptide-specific molecule, which plays an important role in the intracellular secretory pathway. This under the od may be therapeutically important to treat conditions a wide range, associated with hypersecretion and inflammation in humans and animals.

More specifically, the present invention relates to inflammatory cells, which contain mediators of inflammation in one or more granules or vesicles present in the cytoplasm of cells. These cells are subjected to contact with one or more peptides selected from a MANS peptide or an active fragment, each of which is described in detail in this application. Preferably, the contacting of such a peptide with inflammatory cell is carried out by injection of this peptide to an individual suffering from a disease in which inflammatory cells are found in specific tissues or body fluids present in this tissue. After injection of the peptide into the cell or contact of the peptide with the cell specified peptide competes for binding or competitively inhibits binding of a native protein MARCKS with the membrane of intracellular granules or vesicles containing inflammatory mediators. In result of blocking the binding of MARCKS protein to vesicles in inflammatory cells such vesicles in these cells is not transported in the plasma membrane of cells, as it usually happens during stimulation with subsequent ekzoticheskim release the contents of these cells, namely copper is tori inflammation. Thus, the method according to the invention allows to inhibit the transport of vesicles in the plasma membrane of cells, which, in turn, leads to a reduction in release of inflammatory mediators from inflammatory cells. The number of inflammatory mediators that are released from cells during the whole period of time is reduced because the rate of release and the number of released mediators from inflammatory cells depends on the concentration of peptide insertion in inflammatory cells and contacted with these cells.

The advantage of the present invention is that it allows for combination therapy, which includes direct blocking mucus secretion using a unique anti-inflammatory therapy. The advantage of therapy according to the invention compared to current anti-inflammatory therapy, which leads to an overall suppression of the immune system, is that the peptide according to the invention, it is obvious that blocks the secretion of only intracellular components secreted from inflammatory cells. Thus, many aspects of the immune system must function even when the inhibition of inflammatory mediators.

Compounds according to the invention can be adjusted, i.e. to block the release of mediators in the spalania of cells. Such inhibition of release of mediators of inflammation is an attractive tool for the prevention and treatment of various disorders, such as diseases and pathological conditions associated with inflammation. Thus, the compounds according to the invention can be used to treat such conditions. These conditions are diseases of the respiratory tract and chronic inflammatory diseases, including, but not limited to, osteoarthritis, multiple sclerosis, Guillain-Barre syndrome, Crohn's disease, ulcerative colitis, psoriasis, graft versus host and systemic lupus erythematosus. Compounds according to the invention can also be used to treat other disorders associated with elevated activity levels of mediators prosopalgia and enzymes, such as responses to various infectious agents, and various autoimmune diseases such as rheumatoid arthritis, toxic shock syndrome, diabetes, and inflammatory intestinal diseases.

The use of peptides and application of the methods according to the invention enables the treatment of inflammation and therapy, which is based on combination of anti-inflammatory activity of the peptide and its ability to block the secretion of mucus. Diseases, which can be, for example, the chickpeas treatment with the use of the ability of the peptide to block inflammation and mucus secretion, include, but are not limited to, inflammatory intestinal diseases, disorders of the digestive system (i.e., inflammation of the gall bladder, disease of Manatee) and inflammatory diseases of the respiratory tract.

Other Pro-inflammatory mediators are associated with several pathological conditions, which occurs when the influx of neutrophils in inflammation or lesions. It was demonstrated that blocking antibodies can be used to treat the associated with neutrophil tissue damage in acute inflammation (Harada et al., 1996, Molecular Medicine Today 2, 482). In addition to neutrophils, and other cells, which can release mediators of inflammation are other cells, such as basophils, eosinophils, monocytes and lymphocytes, and in this case, therapy can be directed at suppressing the secretion of mediators from these cells. Neutrophils, eosinophils and basophils are a type of granulocyte, i.e. white blood cells, in the cytoplasm which contain granules. Leukocytes synthesize a number of inflammatory mediators that are packaged in cytoplasmic granules and are stored in these granules. Such mediators include, for example, myeloperoxidase [MPO] neutrophils (Borregaard N, Cowland JB Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 1997, 89:3503-3521), peroxidase eosinophils [EPO] and basically major protein MBP] eosinophils (Gleich G J. Mechanisms of eosinophil-associated inflammation. J Allergy Clin. Immunol. 2000, 105:651-663), lysozyme in monocytes/macrophages (Hoff, T., Spencker T., Emmendoerffer A., Goppelt-Struebe M. Effects of glucocorticoids on the TPA-induced monocytic differentiation. J Leukoc. Biol. 1992, 52:173-182, Balboa M A, Saez Y, Balsinde J. Calcium-independent phospholipase A2 is required for lysozyme secretion in U937 promonocytes. J Immunol. 2003, 170:5276-5280), and Grasim in natural cells killer cells (NK) and cytotoxic lymphocytes (Bochan M.R., Goebel W.S., Brahmi Z. Stably transfected antisense granzyme B and perforin constructs inhibit human granule-mediated lytic ability. Cell Immunol 1995, 164:234-239, Gong J. H., Maki g, Klingemann H.G. Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia 1994, 8:652-658, Maki g, Klingemann H.G., Martinson J.A., Y.K. Tam Factors regulating the cytotoxic activity of the human natural killer cell line, NK-92. J. Hematother Stem Cell Res. 2001, 10:369-383, and Takayama H, Trenn G, M.V. Sitkovsky A novel cytotoxic T lymphocyte activation assay. J. Immunol. Methods 1987, 104:183-1907-10). These mediators can be released to the area of injury and may play a role in the development of inflammation and repair, for example, in the lungs and in other organs, due to the infiltration of these cells in the affected area or tissue pathology. Leukocytes release these granules according to the mechanism of exocytosis (Burgoyne R.D, Morgan A. Secretory granule exocytosis. Physiol Rev 2003, 83:581-632, Logan M.R, Odemuyiwa S.O, Moqbel R. Understanding exocytosis in immune and inflammatory cells: the molecular basis of mediator secretion. J Allergy Clin. Immunol. 2003, 111:923-932).

Fat cells are usually not circulating in the bloodstream, and basophils contain cytoplasmic secretory granules that store and can in order to Svobody after activation of cells previously formed mediators of inflammation (anafilaktichesky), such as histamine; proteoglycans, such as heparin sulfate and chondroitin; proteases, such as tryptase, chymase, carboxypeptidase and cathepsin G-like protease; chemotactic factors, cytokines and metabolites of arachidonic acid, which act on the vascular system, smooth muscle, connective tissue, mucous glands and inflammatory cells.

Neutrophils, also known as polymorphonuclear leukocytes (PMN), contain 50-60% of all circulating blood leukocytes. Neutrophils act against infectious agents such as bacteria, fungi, protozoa, viruses, virus-infected cells and tumor cells, which penetrate through the physical barriers of the body at sites of infection or injury. Maturation of neutrophils occurs in six morphological stages: myeloblast, promolast, milotic, metamyelocyte, non-segmented (stab) and segmented neutrophil (functionally active) neutrophil.

In neutrophils, inflammatory mediators are stored in the primary (azurophilic), secondary (specific) and tertiary (gelatinase) granules, and secretory vesicles. Among the various mediators of inflammation, primary (azurophil) granules contain myeloperoxidase (MPO), lysozyme, defensin, protein, increasing permeability of the bacteria (BPI), ELA the pelvis, cathepsin G, cathepsin B, cathepsin D, beta-D-glucuronidase, alpha-mannosidase, phospholipase A2, chondroitin-4-sulfate and proteinase 3 (see, for example, Hartwig J.H; Thelen M, Rosen A, Janmey P.A. Nairn AC, Aderem A. MARCKS is an actin filament crosslinking protein regulated by protein kinase C and calcium-calmodulin. Nature 1992, 356:618-622); secondary (specific) granules contain lysozyme, lactoferrin, collagenase, an activator of complement, phospholipase a2the receptors of complement, for example, CR3, CR4, receptors N-formylmethyl-leucyl-phenylalanine (FMLP), a laminin receptor, cytochrome b558, macrophage-chemotactic factor, histaminase and protein to bind with vitamin B12; and storing small granules contain gelatinase, plasminogen activator, cathepsin In, cathepsin D, beta-D-glucuronidase, alpha-mannosidase and cytochrome b558.

Neutrophil granules contain antimicrobial or cytotoxic substances, neutral proteases, acid hydrolases and pools cytoplasmic membrane receptors. Azurophil granules, in addition to other components, contain myeloperoxidase (MPO), which is a enzyme that plays an important role in the conversion of hydrogen peroxide to hypochlorous acid. This enzyme, together with hydrogen peroxide and a halide cofactor form myeloperoxidase system, which provides effective antimicrobial and citotoksicheskoyj leukocytes.

Defensin that make up 30-50% of all proteins azurophilic granules are small (molecular weight <4000) peptides having strong antimicrobial action, which are cytotoxic for a wide range of bacteria, fungi and some viruses. Their toxicity may be due to the permeability of the membrane of target cells, which are also targets for other canalobre proteins (perforins).

Protein increases the permeability of the bacteria (BPI), is a member of the family of perforin. It is highly toxic to gram-negative bacteria, but not gram-positive bacteria or fungi, and may also neutralize endotoxin, i.e. toxic component of lipopolysaccharide membrane of gram-negative bacterial cells.

Lactoferrine airing not contain iron, and therefore they prevent the growth of oral history of microorganisms, which do not undergo cytolysis and improve permeability of the bacteria to litzkow.

Serine proteases, such as elastase and cathepsin G, hydrolyzing proteins in the membranes of bacterial cells. Substrates of the granulocyte Alatas are cross-linked collagens and proteoglycans, and elastin in blood vessels, ligaments and cartilage. Cathepsin D cleaves Ave is topicana cartilage, while collagenase granulocytes break down collagen type 1 and to a lesser extent collagen type III bone, cartilage and tendons. The cleavage products of collagen have chemotactic activity for neutrophils, monocytes and fibroblasts.

Regulation destructive potential of lysosomal proteases in tissue is mediated by protease inhibitors such as alpha-2-macroglobulin and alpha-1-antiprotease. Such antiprotease present in serum and in synovial fluid. They may function by binding to the active centers of enzymes and seizing these active centers. The imbalance of the system "protease-antiprotease" can play an important role in the pathogenesis of emphysema.

Azurophil granules operate predominantly in the intracellular environment (phagolysosomal vacuoles, where they are involved in cytolysis and the decomposition of microorganisms. Retroperspective granules susceptible to release their extracellular content and play an important role in the development of inflammation. Specific granules are intracellular reservoir for the various components of the plasma membrane, including the cytochrome b (a component of NADPH oxidase, the enzyme responsible for the production of superoxide), receptors for a fragment of the complement iC3b (CR3, CR4), receptors for laminin and formylmethyl-peptide Jamoat racconti. Among other components, these granules are histaminase, which plays a role in the decomposition of histamine; vitaminsvitamin protein, and plasminogen activator, which is responsible for the formation of plasmin and dissociation of C5a from C5.

The important role of neutrophilic granules in the development of inflammation was identified in the research conducted with the participation of several patients with congenital anomalies of these granules. In patients with syndrome Chediak-Higashi, there are serious irregularities in the speed of the formation of the inflammatory response and abnormally high content of lysosomal granules. Congenital syndrome defect specific granules is an extremely rare disease characterized by reduced levels of the inflammatory response and the development of severe bacterial infections of the skin and deep tissues.

Although the mechanisms regulating ekzoticheskuyu secretion of these granules, only partially, however, identified several key molecules involved in this process, including intracellular Ca2+in the transition state (Richter et al. Proc. Natl. Acad. Sci USA 1990, 87:9472-9476, Blackwood et al., Biochem J 1990, 266: 195-200), G-proteins, tyrosine - protein kinase (ROK, and in particular PKC) (Smolen et al., Biochim Biophys Acta 1990, 1052:133-142, Niessen et al., Biochim. Biophys. Acta 1994, 1223:267-273, Naucler et al., Pettersen et al., Chest 2002, 121, 142-150), Rac2 (Abdel-Latif et al., Bood 2004, 104:832-839, Lacy et al., J. Immunol. 2003, 170:2670-2679) and various SNARE, SNAP and VAMP (Sollner et al., Nature 1993, 362:318-324, Lacy, Pharmacol. Ther 2005, 107:358-376).

SNARE proteins (soluble receptor protein to bind to the N-ethylmaleimide) belong to the family of membrane-bound proteins characterized by the presence of alpha-superspiritual domain, called the SNARE motif (Li et al., Cell. Mol. Life Sci. 60:942-960 (2003)). These proteins are divided into v-SNARE and t-SNARE on the basis of their localization in vesicles or membrane of target cells, and other classification scheme, these proteins are divided into R-SNARE and Q-SNARE on the basis of the presence of conservative arginine or glutamic residue in the center of the SNARE motif. SNARE localized on separate membrane compartments of the secretory and endocytotic transport routes and contribute to the specificity of intracellular processes merge membranes. The domain t-SNARE consists of a bundle of 4 spirals with superspiritual structure. The SNARE motif plays a role in the fusion of the two membranes. The SNARE motifs are divided into four classes: homologues syntaxin 1a (t-SNARE), VAMP-2 (v-SNARE) and N - and C-terminal SNARE motifs of SNAP-25. One member of each class can interact with the formation of the SNARE complex. The SNARE motif present in the N-terminal domains of some family members syntaxin, such as syntaxin 1a, which is required for the release of neurotransmitters (Lermanet al., Biochemistry 39: 8470-8479 (2000)), and syntaxin 6, which is present in vesicles endosome transport (Misura et al., Proc. Natl. Acad. Sci. U.S.A. 99:9184-9189 (2002)).

Proteins SNAP-25 (assosiated with synaptosomal protein (25 kDa) are components of SNARE complexes, which may be responsible for the specificity of the fusion of membranes and direct merger by formation of a tight complex (SNARE or crustal complex), which binds synaptic vesicles and the plasma membrane. SNARE constitute a large family of proteins characterized by sequences consisting of 60 residues and is known as the SNARE motifs, which have a high degree of predisposition to the formation of superspiritual structures, and in most cases, are precursors carboxy-terminal transmembrane regions. Synaptic core complex formed by four SNARE motifs (two motives from SNAP-25 and one from synaptobrevin and syntaxin 1), which individually are unstructured, but together form a bundle of four parallel helices. The crystal structure analysis of the crustal complex showed that the spiral beam has a high degree of circling and contains several salt bridges on the surface and layers of the inner hydrophobic residues. Polar layer in the center is such a complex is formed by three glutamine (two from SNAP-25 and one from syntaxin 1) and one arginine (from synaptobrevin) (Rizo et al., Nat. Rev. Neurosci 3:641-653 (2002)). Members of the family SNAP-25 contains a cluster of cysteine residues that can be polimetilenovye to associate with the membrane (Risinger et al., J. Biol. Chem. 268:24408-24414 (1993)).

Neutrophils play an important role in the phagocytosis and destruction of infectious agents. They also limit the growth of some microbes before the beginning of the formation of adaptive (specific) immune responses. Although neutrophils play an important role in protecting the host, but they also lead to the complication of many chronic inflammatory conditions and reperfusion damage during ischemia. Hydrolytic enzymes originating from neutrophils, and inactivated as a result of oxidation inhibitors protease can be detected in biological fluids isolated from a zone of inflammation. Under normal conditions, neutrophils can migrate to sites of infection, without damaging the host tissue. However, sometimes you may experience unwanted damage to host tissue. Such damage can occur by several independent mechanisms. Such mechanisms are premature activation during migration, extracellular release of toxic products in the process of cytolysis of certain microbes, remove the infected or damaged host cells and cell debris as the PE the howl stage remodeling tissue, or inability to blocking of acute inflammatory responses. Reperfusion injury ischemia is associated with an influx of neutrophils into the infected tissue and their subsequent activation. This process can be run under the influence of substances released from the affected host cells, or it can be caused by the formation of superoxide under the action of xanthine oxidase.

In normal conditions, the blood may contain a mixture of normal, premirovany, activated neutrophils and with impaired function of neutrophils. In the area of inflammation are present, mainly by activated neutrophils and neutrophils with impaired function. Activated neutrophils enhance the production of intermediates reactive oxygen (ROI). A subpopulation of neutrophils with increased respiratory outbreak was detected in the blood of people with acute bacterial infection and in patients with acute respiratory distress syndrome (ARDS). This is the neutrophil paradox. Neutrophils aggravate the pathology of this condition, due to the significant influx of these cells into the lungs and associated with him in the tissue damage caused by the action of oxidative and hydrolytic enzymes released from activated neutrophils. Violation of the antimicrobial activity of neutrophils, which PR is leads to deterioration in ARDS, can serve as a protective response in that part of the host organism, which is locally induced by the products of inflammation.

The acute phase of thermal damage is also associated with activation of neutrophils with subsequent total violation of the various functions of neutrophils. Activation of neutrophils by immune complexes in synovial fluid contributes to the pathology of rheumatoid arthritis. Permanent activation of neutrophils may also initiate tumor development, because some ROI formed by neutrophils destroy DNA and stimulate the migration of tumor cells under the action of proteases. Patients suffering from severe burns, was the correlation between the development of bacterial infections and decrease in relative and absolute numbers of neutrophils positive for the production of antibodies and complement receptors. It was also found that the neutrophil oxidants nature oxidize low-density lipoprotein (LDL), which are then more effectively attached to the plasma membrane of macrophages via specific acceptor receptors. The absorption of these oxidized LDL by macrophages initiates atherosclerosis. In addition, bromirovannye neutrophils have been found in people with essential hypertension, Hodgkin's disease, inflammation of the bowel disease, psoriasis, sarcoidosis and septicemia where such premirovanii correlates with high concentrations of circulating in the bloodstream TNF-alpha (cachectin).

Hydrolytic damage to host tissue, and consequently the development of chronic inflammatory conditions can occur if the protective layers of antioxidants and antiprotease depleted. It is believed that the deficit antiprotease responsible for the development of pathologies in enfizeme. Many antiprotease are members of the family of inhibitors of serine proteases (SERPIN). Although the blood is enriched with antiprotease, however, these large proteins can be selectively removed from areas of inflammation, which is caused by adhesion of neutrophils to their targets. Oxidative stress can initiate tissue damage by reducing the concentration of extracellular antiprotease to values below the level required for inhibition of the released proteases. Chlorinated oxidants and hydrogen peroxide can inactivate antiproteases, such as inhibitor alpha-1-protease and alpha-2-macroglobulin, which are endogenous inhibitors of elastase, but at the same time activate latent metalloprotease, such as collagenase, gelatinase which play a role in the subsequent inactivation of antiproteases.

Cytoplasmic components of neutrophils t is the train to cause the formation of specific antibodies against neutrophil cytoplasmic (ANCA), closely associated with the development of systemic vasculitis and glomerulonephritis. ANCA are antibodies directed against the enzymes that are present mainly in azurophilic or primary granules of neutrophils. There are three types of ANCA, which may vary according to the nature of the indirect immunofluorescence assay, produced normal fixed with ethanol neutrophils. Diffuse fine granular cytoplasmic fluorescence (cANCA) normally observed in Wegener's granulomatosis, in some cases, microscopic polyarteritis and syndrome charge-Strauss, and in some cases, when sickle cell and segmental necrotorous glomerulonephritis. Target antigen is typically proteinase 3. Perinuclear fluorescence (pANCA) is observed in many cases microscopic polyarteritis and glomerulonephritis. These antibodies are often directed against myeloperoxidase, and their other targets are elastase, cathepsin G, lactoferrin, lysozyme and beta-D-glucuronidase. The third group, called "atypical" ANCA, includes antibodies, detected by fluorescence neutrophil nuclei and some unusual cytoplasmic patterns, with only a few antigens are targets produce pANCA, and other antigen targets have not yet been identified. ANCA were detected in one third of patients with Crohn's disease. It was reported that the prevalence of ANCA in rheumatoid arthritis and SLE varies considerably, with predominantly observed pANCA and atypical ANCA.

Eosinophils are leukocytes final stage of differentiation, which are present mainly in the layer of tissue beneath the mucous membrane and the recruitment of such leukocytes occurs in areas of specific immune responses, including allergic reactions. The cytoplasm of eosinophils contains large ellipsoidal granules with electronmobility crystalline core and a partially permeable matrix. In addition to these large initial crystalloid granules are granules and the other type, which have a smaller size (small granules) and do not contain crystalline core. Large specific granules of eosinophils contain at least four different cationic protein, which have different biological effects on the cells of host and microbial targets, namely major major protein (MBP), cationic protein of eosinophils (ECP), a neurotoxin, derived from eosinophils (EDN), and peroxidase eosinophils (EPO). Basophils contain approximately one-fourth of all major major proteins present in eosinophils, together with detectivesyme quantities EDN, ECP and EPO. A small number of EDN and ECP are also found in neutral fileh (Gleich G. J. Mechanisms of eosinophil-associated inflammation. J. Allergy Clin. Immunol. 2000, 105:651-663). MBP is obviously not possess enzymatic activity and are highly cationogenic polypeptide, which may have toxic activity caused by interaction with lipid membranes, leading to their destruction. MBP and EPO can act as selective allosteric inhibitors of agonist binding to muscarinic M2 receptors. These proteins may play a role in the dysfunction of the receptor M2 and strengthen mediated by the vagus nerve, bronchostenosis in asthma. EDN can specifically damage the myelin sheath of neurons. Histaminase and various hydrolytic lysosomal enzymes are also present in most of the specific granules of eosinophils. In a small granules of eosinophils among other enzymes are present in arylsulphatase, acid phosphatase, metalloproteinase size of 92 kDa, gelatinase. Eosinophils can produce cytokines, which have a potential for autocrine activity of growth factors for eosinophils and which play a potential role in the generation of acute and chronic inflammatory responses. There are three cytokines that possess the activity of growth factors for eosinophils, namely: granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3 and I-5. Other cytokines produced by human eosinophils may possess activity aimed at the production of acute and chronic inflammatory responses, and these cytokines are IL-1 alpha, IL-6, IL-8, TNF-alpha and transforming growth factors, TGF-alpha and TGF-beta.

Eosinophils contain crystalloid granules, which include MBP, eosinophilic cationic protein, EPO and eosinophil neurotoxin (Gleich, J. Allergy Clin. Immunol. 2000, 105:651-663). Clone 15 human promyelocytic cell line HL-60 can be used to assess the secretion of EPO. This cell line was derived from clone HL-60, which is cultivated at high pH for two months (Fischkoff, Leuk Res 1988, 12:679-686), and then processed oil acid for maintenance of cell differentiation and messages to these cells many of the intrinsic properties of eosinophils in the peripheral blood, including the expression of proteins eosinophil-specific granules (Rosenberg et al., J. Exp. Med. 1989, 170:163-176, Tiffany et al., J. Leukoc. Biol. 1995, 58:49-54, Badewa et al., Exp. Biol. Med. 2002, 227:645-651).

Eosinophils may be involved in hypersensitivity reactions, in particular, by means of two lipid mediators of inflammation, leukotriene4(LTC4) and platelet activating factor (PAF). Both mediator reduce smooth muscle of the respiratory tract, stimulating the secretion of mucus, alter vascular permeability and call the live infiltration of eosinophils and neutrophils. In addition to the direct activity of these eosinophilic mediators, MBP can stimulate the release of histamine from basophils and mast cells, and MBP can stimulate the release of EPO from the fat cells. Eosinophils can serve as a local source-specific lipid mediators, and can also induce the release of mediators from mast cells and basophils. The contents of eosinophilic granules is released after a similar stimulation of neutrophil granules, for example, in the process of phagocytosis opsonizing particles under the action of chemotactic factors. Neutrophil lysosomal enzymes act mainly on the material that is present in phagolysosome, whereas the contents of eosinophilic granules operates mainly in the extracellular structure of the target, such as parasites and inflammatory mediators.

Monocytes and macrophages develop in the bone marrow and undergo the following stages: a stem cell, commitirovannah stem cell, monoblast, promonocyte, monocyte bone marrow monocyte in the peripheral blood and macrophages in the tissues. Differentiation of monocytes in the bone marrow occurs quickly enough (1.5-3 days). In the process of differentiation in the cytoplasm of monocytes are formed pellets, and these pellets, as in neutrophils, can be subdivided at least into two types. Od is ako they are smaller and have a smaller size, than their neutrophilic analogues (azurophil and specific granules). These granules contain the same enzymes.

Associated with the granule enzymes monocytes/macrophages are lysozyme, acid phosphatase and beta-glucuronidase. As a model forin vivoresearch was used by the secretion of lysozyme from U937 cells. This cell line is derived from human histiocytomas lymphoma and was used as a macrophage cell line, which can be activated by various agonists such as PMA (Hoff et al. J. Leukoc. Biol. 1992, 52:173-182; Balboa et al., J. Immunol. 2003; 170:5276-5280, Sundstrom et al., Int. J. Cancer 1976; 17:565-577).

Natural killer cells (NK) and cytotoxic lymphocytes contain potent cytotoxic granules containing perforin, a pore-forming protein, granzyme and lymphocyte-specific serine protease. For example, cell line NK-92 is an IL-2-dependent human cell line isolated from a patient with rapidly progressive non-Hodgkin lymphoma (J.H. Gong, Maki G, Klingemann H.G. Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia 1994, 8:652-658). Cells NK-92 Express high levels of molecules involved in the cytolytic path perforin-granzyme, aimed at cancer cells a wide range (Gong et al, vide infra, and Maki G, Klingemann H.G, Martinson J.A, Tarn Y.K. Factor regulating the cytotoxic activity of the human natural killer cell line, NK-92. J Hematother Stem Cell Res 2001; 10:369-383).

Granzyme are exogenous serine proteases that are released by cytoplasmic granules in cytotoxic T-cell and natural cell-killers. Granzyme can induce apoptosis in virus infected cells and thereby destroy these cells.

Extracellular secretion of a mediator of inflammation (inflammatory mediator) granulocyte (or leukocyte) and extracellular secretion of more than one mediator of inflammation (inflammatory mediator) granulocyte (or leukocyte) is sometimes called here degranulation. In a preferred embodiment of the invention, the release of a mediator of inflammation is the release of the above mediator of granules located inside of granulocyte or leukocyte. The release of a mediator of inflammation, preferably, is a release of the above mediator of inflammation of these granules.

In neutrophils and macrophages after their premirovany proinflammatory agents (stimulants inflammation), such as TNFα, dramatically increases protein synthesis MARCKS, and more than 90% of the total number of new proteins produced in response to TNFα or lipopolysaccharide (LPS), is a protein MARCKS (Thelen M, Rosen A, Nairn AC, Aderem. A. Tumor necrosis factor alpha modifies agonist-dependent responses in human neutrophils by inducing the synthesis and myristoylation of a secific protein kinase C substrate. Proc. Natl. Acad. Sci. USA 1990, 87:5603-5607). Thus, MARCKS may play an important role in the subsequent release of inflammatory mediators in the case that contains granule cells, such as neutrophils and macrophages were stimulated with agonists, and in particular agonists that act through the activation of PKC (Burgoyne et al., Physiol Rev 2003, 83:581-632, Logan et al. J. Allergy Clin. Immunol. 2003, 111:923-932, Smolen et al., Biochim. Biophys. Acta 1990, 1052:133-142, Niessen et al., Biochim. Biophys. Acta 1994, 1223:267-273, Naucler et al., J Leukoc Biol. 2002, 71:701-710).

In one aspect of the invention, the introduction of inhibiting the degranulation of the number described here of MANS peptide or an active fragment into the area of inflammation in an individual, which was the seat of the disease, pathological condition, or injury, or place of introduction of alien bodies, or their combinations, may lead to reduction mediator of inflammation released from infilterate of leukocytes in the area of inflammation, where these cells, preferably, are granulocytes. The introduction of the MANS peptide and/or at least one active fragment can lead to reduction mediator of inflammation released from leukocytes, such as granulocytes, infilterate in the area of inflammation. Inhibiting the degranulation number of MANS peptide or inhibiting the degranulation of the number of its active fragment of I which is sufficient for reducing or inhibiting the level ekzoticheskogo release of inflammatory mediators from granules, contained in inflammatory cells, infilterate in the specified area of inflammation. The efficiency of inhibition of degranulation determine immediately after the introduction of MANS peptide or an active fragment by comparing the percentage of inhibition (i.e. reduction percentage) release of inflammatory mediators from these cells (leukocytes or granulocytes or other inflammatory cells) in relation to the level or amount or concentration of these mediators of inflammation released or produced at about the same time in the absence of MANS peptide and/or in the absence of its active fragment. In addition, the Clinician can determine the degree of reduction of inflammation in the tissue by identifying symptoms and measurements of parameters of inflammation, known as the indicators of the disease, in order to assess whether the number of the MANS peptide and/or its active fragment sufficient or therapeutically effective for such attenuation of inflammation. Amount sufficient for inhibition of degranulation, is the amount that provides a certain percentage reduction mediator of inflammation released from granulocytes at the site of inflammation, where the specified percentage is about 1% - 99%, preferably about 5% to 99%, more preferred is entrusted, about 10% to 99%, even more preferably about 25% to 99%, and most preferably about 50% to 99% of the quantities specified mediator of inflammation released from these cells in the absence of MANS peptide or an active fragment tested in the same conditions.

In one aspect of the invention, the introduction of inhibiting the degranulation of the number of MANS peptide in the area of stimulation of inflammation in an animal, where the area of inflammation was induced by the introduction into it stimulating inflammation of the number of stimulator of inflammation, may lead to reduction mediator of inflammation released from granulocytes stimulated specified stimulator of inflammation in the specified area stimulating inflammation, approximately 1% to 99%, preferably about 5% to 99%, more preferably about 10% to 99%, even more preferably about 25% to 99%, and most preferably approximately 50% - 99% of the quantities specified mediator of inflammation released from these cells in the absence of peptide MANS, but in the presence of identical stimulating inflammation of the amount specified stimulator of inflammation.

In another aspect of the invention, the introduction of inhibiting the degranulation of the number of MANS peptide in the area of stimulation of inflammation in an animal, where the specified area is ü inflammation was induced by the introduction into it stimulating inflammation of the number of stimulator of inflammation, may cause reduction mediator of inflammation released from granulocytes stimulated specified stimulator of inflammation in the specified area stimulating inflammation, 100% of the amount specified mediator of inflammation released from these cells in the absence of peptide MANS, but in the presence of identical stimulating inflammation of the amount specified stimulator of inflammation.

An example of a stimulator of inflammation usedin vitro as described in the examples provided in this application is phorbol-12-myristate-13-acetate (PMA). Macrophage protein-chemoattractant (MCP-1) has almost the same efficiency as C5a, and much greater activity than IL-8, in relation to the degranulation of basophils and causes the release of histamine. The release of histamine can occur after stimulation by chemokines (cytokines-chemoattractant), RANTES and MIP-1.

With regard to basal concentrations of MARCKS peptide that is present in the area of stimulation of inflammation, in the preferred embodiment of the invention, inhibiting the degranulation number of MANS peptide, administered to the animals in the area of stimulation of inflammation, approximately 1-1000000 times, preferably, about 1-100000 times, more preferably, about 1-10000 times, even more preferably, about 1 to 1,000 times, more preferred the equipment, about 1-100 times, and most preferably, about 1-10 times the concentration of the MARCKS peptide in the specified area stimulating inflammation.

In a preferred embodiment of the invention, the granulocytes are present on the surface or inside of the respiratory tract of the animal, and preferably human, and therefore, the MANS peptide is administered by inhalation, for example, pharmaceutical compositions containing the specified MANS peptide, for example, pharmaceutical compositions containing the MANS peptide, and an aqueous solution, where the specified composition is administered in the form of a spray or in the form of a dry powder or a specified composition is injected with insufflator. Can be applied to other methods and devices for introducing a solution or powder by inhalation, such as drops, sprays and nebulizers.

In some embodiments of the invention, the peptide according to the invention can block physiologically important secretory processes, including blocking basal secretory functions. Not limited to any particular theory, the authors present invention believe that the mechanisms of regulation of such basal secretion differ from the mechanisms of regulation of stimulated secretion. Alternatively, for the implementation of the basal mechanisms of secretion may require fewer MARCKS protein than stimulated sec is ecii. Basal secretion can be saved, since all therapies aimed at blocking MARCKS-mediated secretion, and can not overpower all of MARCKS function.

Used herein, the term "nucleotide sequence MARCKS" refers to any nucleotide sequence derived from the gene encoding the protein MARCKS, including, for example, a sequence of DNA or RNA, the DNA sequence of a gene, any transcribed sequence of the RNA sequence RNA pre-mRNA or mRNA transcript, as well as DNA or RNA associated with the protein.

Accurate delivery MARCKS-blocking peptide can also prevent any possible restrictions blocking important secretory processes. Delivery of such agents to the respiratory tract can be easily implemented using the compositions, administered by inhalation. Because such agents can be used to treat inflammatory bowel disease, can be treated and delivery blocking agents in the region of the rectum/colon/small bowel with an enema or suppositories. Intra-articular injection or percutaneous delivery to the inflamed joints can bring relief to patients with arthritic or autoimmune diseases by limiting the secretion from cells localized at the site of inflammation. Injection in which blasty, surrounding the nerves may lead to inhibition of secretion of neurotransmitters, i.e. to block the signals of occurrence of severe pain or uncontrolled spasms of the muscles. Delivery of the peptide for the treatment of inflammatory skin diseases can be easily implemented using different preparations for local administration, known to specialists.

Obviously, MARCKS interacts with actin and myosin in the cytoplasm, and therefore he may have the ability to bind the granules with the cellular contractile apparatus and thereby mediate the further transport of the granules and exocytosis. Secretion of a mediator of inflammation MPO from neutrophils can also be maximized by the activation of PKC and PKG. It is possible that MARCKS is a point of convergence coordinating the actions of these two protein kinases that regulate the secretion of membrane-bound compartments of inflammatory cells (i.e. the secretion of MPO from neutrophils).

In the present invention it was demonstrated that the secretion of a mediator of inflammation lros of dog or human neutrophils increases under the action of competitive activation of PKC and PKG, however, activation of one kinase is not sufficient to induce a maximal secretory response. Enhanced secretory response to PMA was observed in NHBE cells and in stoichiometric who silts, as described in this application, although the magnitude of this response was much less than the magnitude of the response observed in a rat cell line goblet cells. See, above Abdullah et al. In addition, although earlier it was reported that the analogue of cGMP can induce a significant level of secretion of mucin from cultured epithelial cells of the trachea of Guinea pigs (Fischer et al, see above), however it should be noted that this response did not reach significant levels within 8 hours after the start of treatment. Secretory response with such a long lag period (time lag) is unlikely to give direct effect, and, most likely, he will participate in protein synthesisde novoand not in the release already educated and spare cytoplasmic granules.

As mentioned above, the present invention can be used to obtain a pharmaceutical composition. In some embodiments of the invention, the medicinal product is present in the solid pharmaceutical composition, which can be used for oral administration. The solid composition of the present invention, can be prepared with filler, or it may be mixed with a filler and/or divorced in the filler. Consider the solid composition may be encapsulated in a carrier, which may be, e.g. the, capsule, sachet, tablet, paper or other container. If the excipient serves as a diluent, it may be solid, semi-solid, or liquid material which acts as excipient, carrier or medium for the composition.

Various suitable fillers known to specialists in this field, and they can be found on pages 2404-2406 National register of medicines (National Formulary, 19:2404-2406 (2000)), which in its entirety are introduced in the present description. Examples of suitable fillers include, but are not limited to, starches, Arabic gum, calcium silicate, microcrystalline cellulose, methacrylates, shellac, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The composition of the drug product can further include sizing, such as, for example, talc, magnesium stearate and mineral oil; wetting agents; emulsifiers and suspendresume agents; preservatives, such as methyl - and propylhydroxybenzoate; sweeteners or flavours. Can also be used polyols, buffers, and inert fillers. Examples of polyols include, but are not limited to, mannitol, sorbitol, xylitol, sucrose, maltose, glucose, lactose, dextrose, etc. with Suitable buffers include, but are not limited to, phosphate, citrate, tartrate, succinate, etc. Other inert fillers which may be used are fillers known in the art and suitable for use in the preparation of various dosage forms. If necessary, the solid composition can include other components, such as agents that adds volume and/or granulators and other Medicinal products according to the invention can be prepared for fast-acting or slow release of the active ingredient after administration of such products to the patient by methods well known to specialists.

In order to obtain tablets for oral administration, consider the composition according to the invention can be obtained by direct pressing. In this process, active pharmaceutical ingredients can be mixed with solid volatile media, such as, for example, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose derivatives or gelatin, and mixtures thereof, as well as with an antifriction agent such as, for example, magnesium stearate, calcium stearate and waxes based on polyethylene glycol. Then the mixture can be compressed into tablets on a machine with appropriate punches and dies to obtain tablets of the desired size. The operating parameters of this machine can be selected by the specialist. Alternative tablets for oral administration can be obtained by the method of wet granulation. Active medicinal ingredients can be mixed with excipients and/or carriers. Solids can be crushed or sieved to obtain particles of the desired size. In the drug can be added binding agent. The binding agent can be suspended and homogenized in a suitable solvent. The active ingredient and auxiliary agents can also be mixed with a solution of a binding agent. The obtained dry mixture is uniformly moistened with a solution. Hydration usually gives the particles a certain degree of aggregation, and therefore the resulting mass is pressed through a stainless steel sieve with the desired cell size. After that, the mixture is dried in the drying oven with controlled temperature within a certain period of time, necessary to achieve the desired particle size and consistency. The granules are dried mixture is sifted to remove any powder. To this mixture may be added dezintegriruetsja, anti-friction and/or anti-adhesive agents. Finally, the mixture is pressed into tablets on a machine with appropriate punches and dies to obtain tablets of the desired size. The operating parameters of this machine can be selected by the technician.

If you want to get a tablet coatings, obtained as described above, the core tablet can is to be coated with a concentrated solution of sugars or cellulose polymers, which may contain the Arabian gum, gelatin, talc, and titanium dioxide, or lacquer dissolved in a volatile organic solvent or mixture of solvents. For identification of tablets with different active compounds or with different amounts of present active connection to this coverage can be added various dyes. In a specific embodiment of the invention, the active ingredient may be present in the core surrounded by one or several layers, including layers Intercollege coverage.

Can be obtained soft gelatin capsules containing a mixture of active ingredient and vegetable oil. Hard gelatine capsules may contain granules of the active ingredient in combination with a solid volatile media, such as, for example, lactose, saccharose, sorbitol, mannitol, potato starch, corn starch, amylopectin, cellulose derivatives and/or gelatin.

Liquid preparations for oral administration can be prepared in the form of syrups or suspensions, e.g. solutions containing the active ingredient, sugar and a mixture of ethanol, water, glycerol and propylene glycol. If necessary, such liquid preparations may contain one or more of the following components, such as colorants, flavors and saccharin. Can be the e used thickeners, such as carboxymethylcellulose.

If the abovementioned pharmaceutical preparations are used for parenteral administration, such drugs can include sterile aqueous solutions for injection, anhydrous solutions for injection, or both, comprising the composition according to the invention. In the case of obtaining aqueous solutions for injection, the composition according to the invention can be present in the form of a water-soluble pharmaceutically acceptable salt. Preparations for parenteral administration may contain antioxidants, buffers, bacteriostatic and dissolved substances, which reported this drug isotonicity with the blood of the recipient. Aqueous and non-aqueous sterile suspensions can contain suspendresume agents and thickeners. Such preparations may contain in containers designed to store single dose or doses for repeated administration, for example, sealed ampoules and vials. Solutions or suspensions prepared in the form of injections for immediate admission can be obtained from sterile powders, granules and tablets of the previously described type.

This composition may also be prepared so that it could be used for local application (for example, in the form of a cream for application to the skin). Such compositions can in order to contain various fillers, well-known specialists. Suitable fillers can be, but are not limited to, wax-based atilovykh esters, cetyl alcohol, white wax, glycerylmonostearate, propylene glycol, monostearate, methyltert, benzyl alcohol, sodium lauryl sulfate, glycerin, mineral oil, water, carbomer, ethyl alcohol, acrylate adhesives, polyisobutylene adhesives and silicone adhesives.

In a preferred embodiment of the invention, peptide fragments described in table 2 and have a length of at least 4-23 amino acid residue, and amino acid sequence identical to the amino acid sequence of the peptide MANS, where these N-terminal amino acids of these peptides selected from the amino acids present at positions 2-21 peptide sequence MANS (SEQ ID NO:1). More preferably, the peptide fragment has a length of at least 6 amino acids to 23 amino acids. These peptides preferably are acylated at the alpha N-terminal amino acids, and more preferably, they are monitorowanie in the alpha N-terminal position of amino acids.

As illustrated in figure 5, MARCKS fosfauriliruetsa under action is eat protein kinase C (PKC), and then is transported from the membrane into the cytoplasm. In the cytoplasm, PKG, obviously, induces the dephosphorylation of MARCKS (figa, lane 4, and figv). This dephosphorylation is terminated under the action of PKG inhibitor, Rp-8-Br-PET-cGMP (figa, lane 5), suggesting that dephosphorylation, in particular, is a PKG-dependent. In figure 2, NHBE cells were labeled with [32P]orthophosphate, and then they were treated with the indicated reagents. Phosphorylation of MARCKS in response to such processing was evaluated using immunoprecipitation analysis. On figa, 8-Br-cGMP prevents MARCKS phosphorylation induced by PMA, and this effect of 8-Br-cGMP can be blocked under the action of Rp-8-Br-PET-cGMP (PKG inhibitor) or okadaic acid (an inhibitor of PP1/2A). On FIGU, PMA-induced phosphorylation of MARCKS is terminated after treatment of cells 8-Br-cGMP. Track 1 only Wednesday, 8 minutes; lane 2, 100 nm PMA, 3 minutes; lane 3, 100 nm PMA, 3 minutes, and then 1 μm 8-Br-cGMP, 5 min; lane 4, 100 nm PMA, 8 minutes; lane 5, only the environment, 3 minutes, and then 100 nm PMA + 1 μm 8-Br-cGMP, 5 minutes. In Fig. 2C, 8-Br-cGMP-inducirowannoe the dephosphorylation of MARCKS is suppressed by the action of fostriecin depending on concentration.

Obviously, PKG dephosphorylated MARCKS through activation proteinopathy. As illustrated in figa (track 6), akadanova acid at a concentration of 500 nm, while the EU is ü at a concentration which can inhibit PPl and PP2A, blocks PKG-induced the dephosphorylation of MARCKS, suggesting that PKG causes dephosphorylation under the action of activating PPl and/or PP2A. Additional studies using fostriecin and using direct analysis in fosfatazu activity, showed that only PP2A was activated under the action of PKG and was responsible for removing phosphate groups from MARCKS (figs). Obviously, akadanova acid or fostriecin, at concentrations that inhibit PKG-induced the dephosphorylation of MARCKS, reduce the secretion of mucin induced by PMA+8-Br-cGMP or UTP, as shown in figure 3. Figure 3 shows that PP2A is a major component of the path secretion of mucin. The NHBE cells pre-incubated with fostriecin and okadaic acid at the specified concentration (500nm), or one environment within 15 minutes, and then stimulated with PMA (100 nm)+8-Br-cGMP (1 μm) for 15 minutes or UTP (100 μm) for 2 hours. The level of secreted mucin was measured using ELISA. Data are presented as mean ± srcpos. (n=6 at each point), where * denotes significant difference from control (environment) (p<0,05); † indicates significant difference from PMA+8-Br-cGMP-stimulation (p<0,05); and ‡ indicates significant difference from UTP stimulation (p<0,05). Thus, it is obvious that DeVos arilirovaniya MARCKS under the action of PKG-activated PP2A is a major component of the transmission signal, leading to exocytosis Musinovich granules.

To identify the molecular mechanisms by which MARCKS is a link between activation of the kinase with the secretion of mucin, were conducted thorough research of MARCKS phosphorylation in response to activation of the PKC/PKG. As illustrated in figa, PMA (100 nm), probably induces a significant increase (3-4 times) the level of phosphorylation of MARCKS in NHBE cells, and this phosphorylation was attenuated by the action of PKC inhibitor, calphostin C (500 nm). After phosphorylation of MARCKS TransportRule from the plasma membrane to the cytoplasm (Fig. 1B). More specifically, figa shown that activation of PKC leads to phosphorylation of MARCKS in NHBE cells. Cells were labeled with [32P]orthophosphate for 2 hours, and then treated stimulating and/or any abscopal reagents. Phosphorylation of MARCKS in response to the treatment was evaluated by means of thus, as described above. Lane 1, medium (control); lane 2, native, 0.1% of Me2SO; lane 3, 100 nm 4α-PMA; lane 4, 100 nm PMA; lane 5, 100 nm PMA+500 nm, calphostin C; lane 6, 500 nm calphostin C. In Fig. 1B demonstrated that phosphorylated MARCKS is transported from the plasma membrane into the cytoplasm.32P-labeled cells were treated with PMA (100 nm) or the same medium for 5 minutes, and then allocated the membrane and t is tosolini faction. Activation of PKG under the action of 8-Br-cGMP (1 μm), then there is another event activating kinase required for the stimulation of secretion of mucin, did not lead to the phosphorylation of MARCKS, indeed, observed the opposite effect: the MARCKS phosphorylation induced by PMA, stopped under the action of 8-Br-cGMP (figa). This effect of 8-Br-cGMP was not due to suppression of PKC activity, as PMA-induced phosphorylation can be eliminated by the subsequent addition of 8-Br-cGMP in cells (pigv). Therefore, activation of PKG, probably leads to dephosphorylation MARCKS.

Additional research has demonstrated that PKG-induced the dephosphorylation of MARCKS was blocked under the action of 500 nm okadaic acid, an inhibitor of proteinopathy (type 1 and/or 2A (PP1/2A))(figa, track 6). Thus, it is obvious that dephosphorylation is mediated by PPl and/or PP2A. To determine the subtype of the current proteinopathy, additional studies on the phosphorylation was used a new and more specific inhibitor of PP2A, fostriecin (IC50=3,2 nm). As illustrated in figs, fostriecin inhibits PKG-induced the dephosphorylation of MARCKS depending on concentrations (1-500 nm), suggesting that PKG induces dephosphorylation by PP2A activation. To confirm additional activation of PP2A under de is the effect of PKG in NHBE cells was determined by the activity of cytosolic PP1 and PP2A after treatment of cells with reagent 8-Br-cGMP. The PP2A activity was increased approximately 3-fold (from 0.1 to 0.3 nmol/min/mg protein, p<0,01) at concentrations of 8-Br-cGMP, comprising up to 0.1 μm, and the activity of the PPl did not change. The data obtained showed that PP2A can be activated under the action of PKG and is responsible for the dephosphorylation of MARCKS. In accordance with this kind of PP2A activity, obviously, plays a crucial role in the secretion of mucin; and if PKG-induced the dephosphorylation of MARCKS is blocked under the action of okadaic acid or fostriecin, the secretory response to activation of the PKC/PKG or UTP stimulation was improved (figure 3).

MARCKS is associated with actin and myosin in the cytoplasm

Figure 4 illustrates immunoprecipitation analysis with radioactive tagging, in which it was revealed that MARCKS may be associated with two other proteins (approximately 200 and approximately 40 kDa) in the cytoplasm. In figure 4, NHBE cells were labeled with [3H]leucine and [3H]Proline during the night, and membrane and cytosolic fractions were obtained as described under "Experimental procedures". The selected fraction was pre-purified using preimmune control antibodies (6F6). Then the cytosol was divided into two equal fractions and used for thus carried out in the presence of 10 μm of cytochalasin D (Biomol, Plymouth Meeting, Pa.) using the m anti-MARCKS antibody 2Fl2 (lane 2) and preimmune control antibodies 6F6 (lane 3), respectively. Protein MARCKS in the membrane fraction was also analyzed using the thus conducted using antibodies 2Fl2 (track 1). The precipitated protein complex was separated by electrophoresis in 8% polyacrylamide gel with LTOs and visualized using autoradiography using intensifying screens. It is obvious that MARCKS is associated with two cytoplasmic proteins having a molecular weight of about 200 and 40 kDa, respectively. These two MARCKS-related protein was cut from the gel and analyzed using a matrix of laser mass spectrometry desorption ionization/time-of-flight mass spectrometry/internal sequencing of the Protein/DNA Technology Center of Rockefeller University, N. Y.). The data obtained for the mass and sequence of the peptide used to search in the database of proteins with the help of Internet programs ProFound and MS-Fit. The results showed that these proteins are myosin (heavy chain, a second type (A) and actin, respectively. The analysis, conducted using a matrix of laser mass spectrometry desorption ionization/time-of-flight mass spectrometry/internal sequencing showed that these two MARCKS-related protein are myosin (heavy chain, a second type (A) and actin, respectively.

These studies allow us to propose a new paradigm is otnositelno mechanism of signal transmission, regulatory ekzoticheskuyu secretion Musinovich granules of the respiratory tract, and indicate that such a mechanism, obviously, is the first direct evidence of a specific biological function of MARCKS in the physiological process. MARCKS serves as a key molecule mediator regulating the release Musinovich granules from human epithelial cells of the respiratory tract. Obviously, for the secretion of mucin respiratory tract requires dual activation and the synergistic action of PKC and PKG. Activated PKC phosphorylates MARCKS, which leads to the transport of MARCKS from the inner surface of the plasma membrane into the cytoplasm. Activation of PKG, in turn, leads to activation of PP2A, which dephosphorylates MARCKS in the cytoplasm. Because the ability of MARCKS to contact the membrane depends on its state of phosphorylation, such dephosphorylation allows the protein MARCKS again to acquire the ability to bind to the membrane and to join the cytoplasmic membranes Musinovich granules. Through this interaction with actin and myosin in the cytoplasm (figure 4), MARCKS may have the ability to bind the granules with the cellular contractile apparatus and thereby mediate the transport of granules at the periphery of the cells and subsequent release by exocytosis. The wide distribution of MARCKS allow the us to assume the possibility of this or a similar mechanism may regulate the secretion of membrane-bound granules in cells of different types under normal or pathological conditions.

As shown in figure 5, MARCKS may function as a molecular linker via interaction with membrane pellets at its N-terminal domain and binding to actin in the website PSD and thereby to bind the beads with the contractile cytoskeleton for subsequent transport and exocytosis. Figure 5 shows a possible mechanism, indicating that secretion interacts with mucin epithelial (goblet) cells of the respiratory tract and activates two distinct protein kinases, PKC and PKG. Activated PKC phosphorylates MARCKS, which leads to the transport of MARCKS from the plasma membrane into the cytoplasm, whereas PKG activated on the path "nitric oxide (NO) → GC-S → cGMP → PKG", in turn, activates the cytoplasmic PP2A, which dephosphorylates MARCKS. This dephosphorylation stabilizes the accession of MARCKS to the membranes of the granules. In addition, MARCKS interacts with actin and myosin and thereby binds the granules with the cellular contractile apparatus for subsequent transport and release of inflammatory mediators, such as MPO, through exocytosis. Join MARCKS to the granules after its release into the cytoplasm can so the e managed to specific target proteins or some other forms of protein-protein interactions, involving the N-terminal domain of MARCKS. In any case, the MANS peptide or an active fragment containing at least 4 amino acids, have competitive inhibition targeting MARCKS on membrane Musinovich granules and thereby block the secretion.

The present invention also relates to a new method of blocking any cell selecting process through exocytosis, and in particular the release of inflammatory mediators from granules contained in inflammatory cells, in ways stimulation involving MARCKS protein that is a substrate for protein kinase C (PKC), and release the content of membrane-bound vesicles. In particular, the authors present invention has been shown that stimulated the release of a mediator of inflammation, myeloperoxidase, human (6) or dog (7) of neutrophils can be blocked by peptide MANS depending on concentration. In particular, figure 6 shows a selection neutrophils that were stimulated with 100 nm PMA and 10 μm 8-Br-cGMP for secretion of myeloperoxidase (MPO). 100 μm MANS peptide reduces the level of MPO secretion to control levels (*=p<0,05). 10 μm MANS causes a small decrease in the secretion of MPO. 10 or 100 μm of control peptide (RNS) had any effect on the secretion of MPO. 7 shows the selected neutrophils, which was what stimulated with 100 nm PMA and 10 μm 8-Br-cGMP for secretion of myeloperoxidase (MPO). 100 μm MANS peptide reduces the level of MPO secretion to control levels (*=p<0,05). 10 μm MANS causes a small decrease in the secretion of MPO. 10 or 100 μm of control peptide (RNS) had any effect on the secretion of MPO. Thus, the peptide may have a therapeutic application to block the release of inflammatory mediators secreted from infilterate inflammatory cells in all tissues. Many of these released mediators responsible for extensive tissue damage observed in various chronic inflammatory diseases (respiratory diseases, such as asthma, chronic bronchitis and COPD, inflammatory bowel disease, including ulcerative colitis and Crohn's disease, autoimmune diseases, skin diseases such as rosacea, eczema and acne in severe form, and when arthritic and pain syndromes, such as rheumatoid arthritis and fibromyalgia). The present invention can also be applied for the treatment of diseases, such as arthritis, chronic bronchitis, COPD and cystic fibrosis. In accordance with this present invention can be also applied for the treatment of human and animal diseases, in particular diseases affecting horses, dogs, cats and other Pets.

On Fig-12 shows secrecy the MPO in humans and dogs. In all these experiments highlighted neutrophils stimulated with LPS at a concentration of 1×10-6M for 10 minutes at 37°C followed by the addition of stimulants, as indicated on the drawings. Cells were primiraly by lipopolysaccharide (LPS) so that they can respond to the action of secretion.

In one of its variants, the present invention relates to a method of regulation of inflammation in an individual, where the method includes the introduction of a therapeutically effective amount of a pharmaceutical composition containing the MANS peptide or an active fragment. In one aspect of the specified option, the specified active protein fragment MANS contains at least four, and preferably six amino acids. In another aspect of the invention, the said inflammation is caused respiratory diseases, gastrointestinal diseases, skin diseases, autoimmune diseases and pain syndromes. In another aspect of the invention, these respiratory diseases selected from the group consisting of asthma, chronic bronchitis and COPD. In another aspect of the invention, these intestinal diseases selected from the group consisting of ulcerative colitis, Crohn's disease and irritable bowel syndrome. In another aspect of the invention, these skin diseases selected from the group consisting of rosacea, eczema, psoriasis and acne severe. In another aspect of the invention, the specified inflammation caused by arthritis or cystic fibrosis. In another aspect of the invention, the specified individual is a mammal. In addition, in another aspect of the invention, the said mammal is selected from the group consisting of humans, dogs, horses and cats. In another aspect of the invention, the specified stage of introducing selected from the group consisting of local administration, parenteral administration, rectal administration, intra-lungs injection, intranasal administration, administration by inhalation and oral administration. In another aspect of the invention, the specified internal-lung introduction selected from the group consisting of injection and spray, insufflator, nebulizer with dosing valve and the nebulizer.

In another embodiment, the present invention relates to a method of regulating cellular secretory process in the individual, where this method includes the introduction of a therapeutically effective amount of a pharmaceutical composition containing at least one connection involving the MANS peptide or an active fragment, which regulate the release of a mediator of inflammation in an individual. In one aspect of the specified option, the specified active protein fragment MANS contains at least four, and preferably six amino acids In another aspect of the invention, specified by regulation of the cellular secretory process is blocking or weakening of the cellular secretory process. In another aspect of the invention, the specified release of a mediator of inflammation is called respiratory diseases, gastrointestinal diseases, skin diseases, autoimmune diseases and pain syndromes. In another aspect of the invention, these respiratory diseases selected from the group consisting of asthma, chronic bronchitis and COPD. In another aspect of the invention, these intestinal diseases selected from the group consisting of ulcerative colitis, Crohn's disease and irritable bowel syndrome. In another aspect of the invention, these skin diseases selected from the group consisting of rosacea, eczema, psoriasis and acne severe. In another aspect of the invention, the specified inflammation caused by arthritis or cystic fibrosis. In another aspect of the invention, the specified individual is a mammal. In another aspect of the invention, the said mammal is selected from the group consisting of humans, dogs, horses and cats. In another aspect of the invention, the specified stage of introducing selected from the group consisting of local administration, parenteral administration, rectal administration, intra-lungs injection, intranasal, injection and by whom galali and oral administration. In another aspect of the invention, the specified internal-lung introduction selected from the group consisting of injection and spray, insufflator, nebulizer with dosing valve and the nebulizer.

In another embodiment, the present invention relates to a method of reducing inflammation in an individual, where the method includes the introduction of a therapeutically effective amount of a compound that inhibits MARCKS-associated release of inflammatory mediators, where the specified release of mediators of inflammation in the individual is reduced compared to the release, nablyudaemym in the absence of such introduction. In one aspect of this option, the specified connection is at least one active fragment of the protein MARCKS. In another aspect of the invention, the specified active fragment has a length at least four, and preferably six amino acids. In another aspect of the invention, the specified connection is the MANS peptide or an active fragment. In another aspect of the invention, the specified connection is an antisense oligonucleotide directed against the coding sequence of the MARCKS protein or its active fragment. In another aspect of the invention, the specified active fragment has a length at least four, and preferably six amino acids.

In his other Varian is e, the present invention relates to a method of reducing inflammation in an individual, where the method includes the introduction of a therapeutically effective amount of pharmaceutically active composition comprising a compound, an inhibitory MARCKS-associated release of inflammatory mediators, where the specified inflammation of the individual is reduced compared to the inflammation observed in the absence of such introduction. In one aspect of this option, the specified connection is active fragment of the protein MARCKS. In another aspect of the invention, the specified active fragment has a length at least four, and preferably six amino acids. In another aspect of the invention, the specified connection is the MANS peptide or an active fragment. In another aspect of the invention, the specified connection is an antisense oligonucleotide directed against the coding sequence of the MARCKS protein or its active fragment. In another aspect of the invention, the specified active fragment has a length at least four, and preferably six amino acids. The present invention also relates to compositions containing one or more peptides MANS or its active fragments, and to its use in the treatment by inhibiting the release of inflammatory mediators from granules or vesicles inflammatory the cells.

In another embodiment, the present invention relates to a method of reducing or inhibiting inflammation in an individual, where the method includes the introduction of a therapeutically effective amount of at least one peptide comprising the MANS peptide or an active fragment, which are effective for inhibiting or suppressing the release of mediators of inflammation in the area of inflammation. In one aspect of the specified option, the specified active fragment has a length at least four, and preferably at least six amino acids. In another aspect of the invention, these inflammatory mediators produced by cells selected from the group consisting of neutrophils, basophils, eosinophils, monocytes and leukocytes. Preferred cells are leukocytes, preferred cells are granulocytes, and even more preferred are neutrophils, basophils, eosinophils or their combination. In another aspect of the invention, the specified agent is administered orally, parenterally, into the cavity of the body, rectally or through the respiratory tract. In another aspect of the invention, the composition also contains a second molecule selected from the group consisting of antibiotics, antiviral compounds, antiparasitic compounds, anti-inflammatory connection is possible and immunosuppressant.

An active fragment of the MANS peptide can be selected from the group consisting of the peptides described in table 1. As described in this application, such peptides may include, but not necessarily, chemical molecules at the N-terminal and/or C-terminal amino acids.

In another aspect of the invention, the methods described in this invention can be carried out by applying or introducing combinations of the peptides described in table 1 of the present invention, that is, application or administration of one or more of these peptides. In the ways described here, preferably, use or enter one peptide.

In response to the activation of protein kinase C (PKC) under the action of the stimulator of inflammation, degranulation of cells selected from the group consisting of neutrophils, eosinophils, monocytes/macrophages and lymphocytes, can be attenuated by pre-incubation and co-incubation with the peptide, identical to the N-terminal region of MARCKS protein, where the specified peptide selected from the group of peptide fragments MANS, described in table 1. Although the course of treatment and concentration of cells of different types can vary, however, in all cases, the peptide MANS will cause the weakening of the PKC-induced degranulation.

This invention will be demonstrated below, in some examples, the only goal is the beautiful illustrations, and these examples should not be construed as limiting the scope of the invention.Examples

Methods and materials

Immunoprecipitation analysis with radioactive tagging

When tagging [32P]phosphate, the cells were pre-incubated for 2 hours in a modified method of Dulbecco environment Needle that does not contain phosphate and containing 0.2% bovine serum albumin, and then were labeled with 0.1 MCI/ml of [32P]orthophosphate (9000 CI/mmol, PerkinElmer Life Sciences) for 2 hours. For labeling of [3H]myristic acid or3N-amino acids, the cells were incubated overnight in medium containing 50 µci/ml [3N]-myristic acid (49 CI/mmol, PerkinElmer Life Sciences) or 0.2 MCI/ml of [3H]leucine (159 CI/mmol, PerkinElmer Life Sciences) plus 0.4 MCI/ml of [3H]Proline (100 CI/mmol, PerkinElmer Life Sciences). After labeling, the cells were treated stimulating reagents for 5 minutes. If you used the inhibitor, the cells were pre-incubated with the inhibitor for 15 minutes, and then subjected to stimulation. After treatment, cells were subjected to lysis in a buffer containing 50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1 mm EDTA, 10% glycerol, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl, 1 mm benzamidine, 10 μg/ml of pepstatin A and 10 μg/ml leupeptin. Using trichloroacetic acid precipitation and scintillation reading the can is about to determine the effectiveness of radioactive labeling in every culture. Immunoprecipitation MARCKS protein was carried out by the method described Spizz and Blackshear, using cell lysates containing equal number of pulses/min Spizz et al., J. Biol. Chem. 271, 553-562 (1996). Precipitated proteins were separated by electrophoresis in 8% polyacrylamide gel with LTOs and visualized using autoradiography. In this analysis, we used antibody against human MARCKS (2Fl2) and preimmune antibody as a control (6F6).

To assess MARCKS or MARCKS-related protein complexes in different subcellular fractions of radioactively labeled and treated cells are scraped in buffer for homogenization (50 mm Tris-HCl (pH 7.5), 10 mm NaCl, 1 mm EDTA, 1 mm phenylmethylsulfonyl, 1 mm benzamidine, 10 μg/ml of pepstatin A, 10 μg/ml leupeptin), and then destroyed by nitrogen cavitation (800 psi for 20 minutes at 4°C). Cell lysates were centrifuged at 600×g for 10 minutes at 4°C to remove nuclei and intact cells. Supernatant obtained after removal of the cores were divided into membrane and cytosolic fractions by ultracentrifugation at 400000×g for 30 minutes at 4°C. the Membrane sediment was solubilizers in the buffer for lysis by treatment with ultrasound. Then carried out immunoprecipitation, as described above.

MARCKS-associated peptides

Peptides with monitorowanie N-to Navoi sequence (MANS) and randomized N-terminal sequence (RNS) was synthesized in the laboratory Genemed Synthesis, Inc. (San Francisco, Calif.), and then was purified using liquid chromatography high pressure (purity >95%) was confirmed using mass spectroscopy, where it was shown that each individual peak had a molecular mass. The MANS peptide consists of a sequence that is identical to the first 24 amino acids MARCKS, i.e. monitorowanie N-terminal region that mediates embedding MARCKS in membranes, MA-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1 (where MA represents the N-terminal meritorious chain)). The corresponding control peptide (RNS) has the same amino acid composition as the MANS peptide, but its sequence contains amino acids that are located at random, MA - GTAPAAEGAGAEVKRASAEAKQAF (SEQ ID NO:232). The presence of hydrophobic myristate molecules in these synthetic peptides increases their ability to penetrate the plasma membrane, which allows the cells to easily absorb these peptides. To determine the effect of these peptides on the secretion of mucin cells pre-incubated with peptides for 15 minutes and then added secretion, and then evaluated the secretion of mucin by using ELISA.

The antisense oligonucleotides

Antisense oligonucleotide MARCKS and its corresponding control oligonucleotide was synthesized in the laboratory Biognostik GmbH (Gottingen, Germany). Cells NHBE obrabatyvali μm antisense or control oligonucleotide for a maximum of 3 days (in the presence of 2 μg/ml lipofectin within the first 24 hours). The cells are then incubated with secretagogue, and secretion of mucin was estimated using ELISA. Of the treated cells was isolated full-RNA and protein. MARCKS mRNA was assessed using Northern hybridization according to standard procedures using human MARCKS cDNA as a probe. The MARCKS protein level was determined by Western blot analysis using purified anti-MARCKS IgGl (clone 2Fl2) as the "first" of the detecting antibody.

Transient transfection

The domain of the site of phosphorylation (PSD) MARCKS contains PKC-dependent phosphorylation sites and a site that communicates with the actin filament. For design PSD deletional MARCKS cDNA generated two fragments flanking sequence PSD (encoding 25 amino acids), using polymerase chain reaction, and then ligated by Xhol site, which was attached to the 5'-ends of the oligonucleotide primers, designed for polymerase chain reaction. The obtained mutated cDNA and cDNA MARCKS wild-type embedded in the expression vector mammalian pcDNA4/TO (Invitrogen, Carlsbad, Calif.). Selected recombinant constructs was confirmed by hydrolysis restricteduse enzymes and DNA sequencing.

HBEl is a transformed papillomaviruses human bronchial EP is telelog cell line, able to secrete mucin in its cultivation on the phase boundary of the air/liquid. Transfection of cells HBEl was performed using transfairusa reagent Effectene (Qiagen, Valencia, Calif.) in accordance with the manufacturer's instructions. Briefly, differentiated cells HBEl grown on the phase boundary of the air/liquid, subjected to dissociation by trypsin/EDTA and again were seeded into a 12-hole tablets for culturing cells at a density of 1×105cells/cm2. After incubation overnight, the cells were transferrable MARCKS cDNA wild-type cDNA MARCKS, not containing PSD or vector DNA. Cells were cultured for 48 hours for gene expression, and then were treated with secretions and measured the level of secretion of mucin by using ELISA. All procedures transfection was carried out in the presence of the plasmid pcDNA4/TO/lacZ (Invitrogen) (ratio of DNA 6:1, just 1 µg DNA, the ratio of DNA to Effectene reagent = 1:25) for monitoring changes in the efficiency of transfection. The results obtained did not reveal any significant differences in beta-galactosidase activities of cell lysates isolated from transfected cells, which indicates that the various DNA constructs have similar transfection efficiency (data not shown).

Analysis proteinopathy activity

Activity PPl and PP2A from Eraly using system analysis proteinopathy activity (Life Technologies, Inc.), well-known specialists, with minor modifications. Huang et al. Adv. Exp. Med Biol. 396, 209-215 (1996). Briefly, NHBE cells were treated with 8-Br-cGMP or only medium for 5 minutes. The cells are then scraped in buffer for lysis (50 mm Tris-HCl (pH 7,4), of 0.1% beta-mercaptoethanol, 0.1 mm EDTA, 1 mm benzamidine, 10 μg/ml of pepstatin A, 10 μg/ml leupeptin) and subjected disruptive under the action of ultrasound for 20 seconds at 4°C. Cell lysates were centrifuged, and supernatant retained for analysis on fosfatazu activity. This analysis was performed using32P-labeled phosphorylase And the substrate. Level redundant32Piwas determined by scintillation counting. The protein concentration in each sample was determined using analysis of Bradford. The PP2A activity was expressed as total fosfatazu activity of the sample minus the residual activity in the presence of 1 nm okadaic acid. The PPl activity was expressed as the difference between the residual activity in the presence of 1 nm and 1 μm okadaic acid, respectively. Proteinopathies activity was recorded as nmol Pireleased / min/mg total protein.

Analysis of cytotoxicity

All reagents used for the treatment of NHBE cells, was evaluated for cytotoxicity by determining the level of a General release of lactate-d is hydrogenase of cells. The analysis was carried out using a set of Promega Cytotox 96 in accordance with the manufacturer's instructions. All experiments were carried out using reagents in recitations concentrations.

Statistical analysis

The obtained data were analyzed for statistical significance using one-way analysis of variance with amended Bonferroni applied after such analysis (post-test). Differences between treatment groups were considered significant when p<0,05.

The allocation of PMN from the blood of dogs

Stage selection PMN include collection of 10 ml antikoagulyantnoe blood in ACD solution. Then 5 ml of blood was applied layers of 3.5 ml of medium for selection PMN, so that the specified environment to highlight PMN (IM) was maintained at room temperature (RT). After that, the blood was centrifuged at room temperature for 30 minutes, 550×g at 1700 rpm The bottom white strip was transferred to a conical centrifuge the 15 ml tube (CCFT). Then added 2V HESS with 10% fetal bovine serum (PBS) and centrifuged at room temperature for 10 min, 400×g at 1400 rpm then the precipitate resuspendable in 5 ml of 1-1ESS with PBS. The cell suspension was added to 50 ml CCFT containing 20 ml ice 0,88% NH4Cl, and the tube turned 2-3 times. The resulting product was centrifuged within 10 m of the nut, 800×g at 2000 rpm, and then was aspirated and resuspendable in 5 ml of HBSS with FBS. The drug was analyzed by counting cells and cytocentrifugation, preferably cells of whole blood, which should be 109-1011cells, and PMN, which should be 2-4×107cells. In General, see Wang et al., J. Immunol, "Neutrophil - induced changes in the biomechanical properties of endothelial cells: roles of ICAM-I and reactive oxygen species" 6487-94 (2000).

Colorimetric enzymatic analysis of MPO-activity.

The samples were analyzed for MPO-activity in 96-well round-bottom microtiter tablets using the kit sandwich ELISA (R & D Systems, Minneapolis, Minn.). Briefly, 20 microliter sample was mixed with 180 Microlitre substrate mixture containing 33 mm potassium phosphate, pH 6,0, 0,56% Triton X-100, 0,11 mm hydrogen peroxide, and 0.36 mm dihydrochloride O-dianisidine in each well of microtiter plate. Final concentration of the analytical mixture was as follows: 30 mm potassium phosphate, pH of 6.0, 0.05% of Triton X-100, 0.1 mm of hydrogen peroxide and 0.32 mm dihydrochloride O-dianisidine. After mixing the analytical mixture incubated at room temperature for 5 minutes, and the activity of the enzyme MPO was determined on a spectrophotometer at a wavelength of 550 nanometers. The samples were analyzed in duplicate.

Example 1

The study of the secretion of mediators of vocal the tion

Were used leukocytes four different types or models, which secrete specific content of the granules in response to induced turbolover ester activation of PKC. Neutrophils were isolated from human blood and were evaluated byin vitro MPO release by these cells. Also evaluated the release of membrane-bound mediators of inflammation from commercially available human leukocyte cell lines. To assess the secretion of EPO used the clone 15 human promyelocytic cell line HL-60 (Fischkoff SA Graded increase in probability of eosinophilic differentiation of HL-60 promyelocytic leukemia cells induced by culture under alkaline conditions. Leuk Res 1988, 12:679-686, H.F. Rosenberg, Ackerman, S. J., D.G. Tenen Human eosinophil cationic protein: molecular cloning of a cytotoxin and helminthotoxin with ribonuclease activity. J. Exp. Med. 1989, 170:163-176, Tiffany H.L., Li F, H.F. Rosenberg Hyperglycosylation of eosinophil ribonucleases in a promyelocytic leukemia cell line and in differentiated peripheral blood progenitor cells. J. Leukoc. Biol. 1995, 58:49-54, Badewa A.P., C.E. Hudson, Heiman A.S. Regulatory effects of eotaxin, eotaxin-2, and eotaxin-3 on eosinophil degranulation and superoxide anion generation. Exp. Biol. Med. 2002, 227:645-651). To assess the secretion of lysozyme used a cell line monocytic leukemia U937 (Hoff, T., Spencker T., Emmendoerffer A., Goppelt-Struebe M. Effects of glucocorticoids on the TPA-induced monocytic differentiation. J. Leukoc. Biol. 1992, 52: 173-182, M.A. Balboa, Y. Saez, J. Balsinde Calcium-independent phospholipase A2 is required for lysozyme secretion in U937 promonocytes. J. Immunol. 2003, 170:5276-5280, Sundstrom C, Nilsson K. Establishment and characterization of a human histiocytic lymphoma cell line (U-937). Int. J. Cancer 1976, 17:565-577). To assess vysvobozhdeny is granzyme used lymphocytic cell line natural killer cells NK-92 (J.H. Gong, Maki G, Klingeniann H.G. Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia 1994, 8:652-658, Maki G, Klingemann H.G, Martinson J.A, Y.K. Tam Factors regulating the cytotoxic activity of the human natural killer cell line, NK-92. J. Hematother Stem Cell Res 2001, 10:369-383, Takayama H, Trenn G, M.V. Sitkovsky A novel cytotoxic T lymphocyte activation assay. J Immunol Methods 1987, 104:183-190). In all cases, cells were pre-incubated with different concentrations of synthetic peptide identical to the 24 amino acid N-terminal MARCKS (peptide MANS with monitorowanie N-terminal sequence; MA-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1), where MA is myristoyl attached to the N-terminal amine of the peptide through an amide bond), or control missense peptide (RNS: peptide with a randomized N-terminal sequence, MA-GTAPAAEGAGAEVKRASAEAKQAF, SEQ ID NO:232), the sequence of which consists of the same 24 amino acids, but located in an arbitrary order where a specified sequence less than 13% identical to the sequence of the peptide MANS. Alternatively, the cells were pre-treated by one of the synthetic truncated peptides listed below in table 3.

In the cells of each type of Le MANS, but not RNS reduces the release of inflammatory mediators, depending on the concentration. The observation time of this release can be 0.5-3.0 hours. The obtained results correspond to the results obtained for the N-to Navoi region of MARCKS protein, involved in the intracellular pathways that lead to degranulation of leukocytes.

The allocation of human neutrophils

These studies were conducted with the permission of the Regulatory authorities that oversee research involving human subjects (IRB). Human neutrophils were isolated as described above (see Takashi S, OkuboY, Horie S. Contribution of CD54 to human eosinophil and neutrophil superoxide production. J. Appl. Physiol. 2001, 91:613-622) with minor modifications. Briefly, heparinized venous blood was obtained from normal healthy volunteers were diluted with medium RPMI-1640 (Cellgro, Mediatech, Inc., Herndon, VA) in the ratio of 1:1, was applied on histopa (with a density of 1.077 g/ml, Sigma-Aldrich Co., St. Louis, MO) and centrifuged at 400×g for 20 minutes at 4°C. the Supernatant and mononuclear cells at the interface was carefully removed and the erythrocytes in the sediment were subjected to lysis in chilled distilled water. Selected granulocytes twice washed with balanced salt solution Hanks (HBSS) and resuspendable in HBSS on ice. Neutrophils used in the experiments had a purity >98%, where the admixture of eosinophils was <2%, and the viability of neutrophils was >99%, as determined by exclusion Trifanova blue.

Measurement of the activity of MPO released by neutrophils

To measure the release of MPO, peeled celoveceskoe neutrophils, suspended in HBSS, were divided into aliquots at a density of 4×106cells/ml in 15 ml test tubes and pre-incubated with 50 or 100 μm MANS, RNS or one of the peptides according to the invention for 10 minutes at 37°C. the cells are Then stimulated with 100 nm of phorbol-12-myristate-13-acetate (PMA) during the period of time up to 3 hours. Control standard (PMA-standard) was created with the use of purified human neutrophils suspended in HBSS and divided into aliquots at a density of 4×106cells/ml in 15 ml tubes, where these cells were stimulated with 100 nm of phorbol-12-myristate-13-acetate (PMA) in the absence of the test peptide for the same period of time. The reaction was completed by keeping the tubes on ice and centrifugation at 400×g for 5 minutes at 4°C.

The MPO activity in the cell supernatant were analyzed using tetramethylbenzidine (TMB) in accordance with the previously developed technology (Abdel-Latif D, Steward M, Macdonald DL, Francis GA., Dinauer MC, Lacy P. Rac2 is critical for neutrophil primary granule exocytosis. Blood 2004, 104:832-839). Briefly, 100 µl of the TMB substrate solution was added to 50 μl of cell supernatants or standard human MPO (EMD Biosciences, Inc., San Diego, CA) in 96-well-microplate, and then incubated at room temperature for 15 minutes. The reaction was completed by adding 50 μl of 1M H2SO4and optical is latest read at 450 nm on a spectrophotometric microplate reader (VERSA max, Molecular Devices, Sunnyvale, CA).

Research culture of cells

Cell lines human leukocyte three types, and in particular the clone 15 human promyelocytic cell line HL-60, monocytic cell line U937 and lymphocytic cell line natural killer cells NK-92 bought in the American type culture collection (ATCC, Rockville, MD). Cells of clone 15 HL-60 (ATCC CRL-1964) maintained in medium consisting of RPMI medium 1640 with L-glutamine, which has been added 10% thermoinactivation fetal bovine serum (Gibco, Invitrogen Co., Carlsbad, CA), 50 IU/ml penicillin, 50 μg/ml streptomycin, and 25 mm HEPES buffer, pH 7.8, at 37°C in atmosphere containing 5% CO2. Final differentiation into cells eosinophil-like phenotype was performed by culturing cells at a density of 5×105cells/ml in the above medium containing 0.5 mm butyric acid (Sigma-Aldrich Co.), within 5 days as described previously (Tiffany HL, Li F, Rosenberg HF. Hyperglycosylation of eosinophil ribonucleases in a promyelocytic leukemia cell line and in differentiated peripheral blood progenitor cells. J Leukoc. Biol. 1995, 58:49-54, Tiffany H.L, Alkhatib G, Combadiere C, Berger E.A, Murphy P.M. CC chemokine receptors 1 and 3 are differentially regulated by IL-5 during maturation of eosinophilic HL-60 cells. J Immunol. 1998, 160:1385-1392). The U937 cells (ATCC CRL-1593.2) were cultured at 37°C in an atmosphere with 5% CO2in a complete medium consisting of RPMI medium 1640 with L-glutamine, which has been added 10% FBS, 50 IU/ml penicillin and 50 µg/ml streptomycin the and. Cells NK-92 (ATCC CRL-2407) supported in the environment of alpha-MEM (Sigma-Aldrich Co.), to which was added 20% FBS, 100 u/ml interleukin-2 (IL-2) (Chemicon International, Inc. Temecula, CA), 5×10-5M 2 - mercaptoethanol, 50 IU/ml penicillin and 50 µg/ml streptomycin at 37°C in atmosphere containing 5% CO2. The morphology of the cells was estimated by the method of dyeing according to the Wright-Giemsa. Viability collected for the experiment, cells were assessed by exclusion Trifanova blue and used a population of cells with a viability of >95%.

Incubation of cells for analysis on the degranulation

Clone 15 cells HL-60 cells, U937 cells and NK-92 was washed and resuspendable at a density of 2.5×106cells/ml in medium RPMI-1640 containing no phenol red (Cellgro, Mediatech, Inc.), for all analyses on degranulation. Aliquots of the cells in 15 ml test tubes pre-incubated with indicated concentrations MANS, RNS or test peptide for 10 min at 37°C. the cells are Then stimulated by PMA during the period of time up to 2 hours. Control standard (PMA-standard) was created for each cell type, namely clone 15 cells HL-60, U937 cells and cells with NK-92, respectively, which were washed and resuspendable at a density of 2.5×106cells/ml in medium RPMI-1640 containing no phenol red, and then stimulated with PMA in the absence of MANS, RNS or test peptide during the same time period. The reaction was completed by keeping the tubes on ice and centrifugation of the cells at 400×g for 5 minutes at 4°C.

For level measurement of the released MPO from neutrophils and released lysozyme from U937 cells by the authors of the present invention was carried out quantitative assessment of the level secretion using human MPO and egg protein ovalbumin as standards, respectively. To quantify the level of EPO released from clone 15 cells HL-60 and released granzyme cells from NK-92, standards are not used. And therefore measured the levels of EPO release and granzyme and their intracellular levels (lysed cells), and these levels of secreted EPO and granzyme expressed as a percentage of the total level of each enzyme (level of intracellular and secreted enzyme). To measure the level of intracellular EPO in clone 15 cells HL-60 and intracellular granzyme in cells NK-92 was taking appropriate aliquots of cells lysed with 0.1% Triton X-100, in order to quantify the intracellular protein granules described below. To minimize variability between cultures, each treatment was expressed as a percentage of the control.

Measurement of the release of EPO cells HL-60

The activity of EPO released clone 15 cells HL-60, were analyzed using TB in accordance with the previously developed technology (Lacy P, Mahmudi-Azer S, Bablitz B, Hagen S.C, Velazquez J.R, Man S.F, Moqbel R. Rapid mobilization of intracellular stored RANTES in response to interferon-gamma in human eosinophils. Blood 1999, 94:23-32). Thus, 100 μl of TMB substrate solution was added to 50 ál (ál = microliter) sample in 96-well-microplate, and then incubated at room temperature for 15 minutes. The reaction was completed by adding 50 µl of 1,0M H2SO4and the optical density was read at 450 nm on a spectrophotometric microplate reader. The number Sekretareva EPO was expressed as a percentage of the total amount of enzyme present in the same number of cells lysed with Triton X-100.

Measurement of secretion of lysozyme by monocytes

The level of lysozyme secreted by U937 cells, was measured using spectrophotometric analysis as described previously (Balboa MA, Saez Y, Balsinde J. Calcium-independent phospholipase A2 is required for lysozyme secretion in U937 promonocytes. J. Immunol 2003, 170:5276-5280) with minor modifications. Thus, 100 μl of sample was mixed with 100 μl of the suspensionMicrococcus lysodeikticus(Sigma-Aldrich Co.) (0.3 mg/ml in 0.1 M phosphate-sodium chloride buffer, pH 7.0) in 96-well-microplate. The decrease in optical density at 450 nm was measured at room temperature. A calibration curve was built using lysozyme hen egg white (EMD Biosciences, Inc.) as a standard.

Measurement of secretion of granzyme NK-cells

At over granzyme, secreted by cells in NK-92, was measured by assessing the hydrolysis dibenzylamino ether Nα-benzyloxycarbonyl-L-lysine (BLT), essentially as described previously (Takayama H, Trenn G, M.V. Sitkovsky A novel cytotoxic T lymphocyte activation assay. J. Immunol Methods 1987, 104:183-190). Briefly, 50 μl of supernatant was transferred into a 96-well plate, and to this supernatant was added to 150 μl of the solution BLT (0.2 mm BLT, EMD Biosciences, Inc., and 0.22 mm DTNB, Sigma-Aldrich Co.) in phosphate buffered saline (PBS, pH of 7.2). The optical density at 410 nm was read after incubation for 30 minutes at room temperature. The results were expressed as a percentage of total cellular enzyme that is present in the same number of cells lysed with Triton X-100.

Statistical analysis

The statistical significance of differences between the various treatment groups was evaluated using one-way ANOVA. Values of P<0.05 is considered significant.

Inhibition of MPO release from human neutrophils

It was found that after 30 minutes, PMA at a concentration of 100 nm (stimulator of the release of inflammatory mediators) increased the level of MPO release from human neutrophils by approximately three times compared with the control level, and after 3 hours, the release rate increased approximately 5-6 times. After 30 minutes, we measured MPO activity, where the activity of the MPO in the absence of PMA, and in the absence of PMA + MANS, RNS or test peptide relative to the control was taken as 100%, and the activity of MPO in the presence of PMA-model was approximately 275%, the activity of MPO in the presence of PMA + 50 μm MANS was approximately 275%, and the activity of MPO in the presence of 100 μm MANS was approximately 305%. Thus, the MANS peptide was not detected detected effect after 30 minutes. However, after 1 hour, MANS at higher concentration (100 μm) was found significant inhibitory effect (about 260% of control) or caused approximately 25%reduction in release of MPO compared with the level in the presence of PMA-standard (which the measurement was approximately 340% of control). As it was measured, 50 µm sample MANS found the activity of MPO approximately 290% of control, or approximately 15%reduction relative to the activity in the presence of PMA-standard. After 2 hours and continuously for 3 hours, the MANS peptide significantly reduced the MPO activity depending on the concentration. After 2 hours, the activity of MPO in the presence of a control PMA-model was approximately 540% of control; 50 μm MANS (measurements approximately 375% of control) caused an approximately 30%reduction in release of MPO compared to the release rate in the presence of a control PMA-standard and 100 μm MANS (measurements approximately 295% of control) you yval approximately 45%reduction in the release of MPO compared to the release in the presence of a control PMA-standard. After 3 hours, the activity of MPO in the presence of a control PMA-model was approximately 560% of control; 50 μm MANS (measurements approximately 375% of control) caused an approximately 33%reduction in the release of MPO compared with control in the presence of a control PMA-standard and 100 μm MANS (measurements about 320% of control) caused an approximately 40%reduction in the release of MPO compared with control in the presence of a control PMA-standard. The RNS peptide had no effect on PMA-induced release of MPO in any period or under any of the tested concentrations. The data presented in the table below were obtained for a concentration of 100 μm of the tested peptides, which were incubated for two hours with 100 nm PMA.

Inhibition of release of EPO from cell HL-60

The EPO activity in the supernatant of clone 15 cells HL-60 was significantly increased after 1 and 2 hours after PMA-stimulation. After 1 hour, the activity of the EPO in relation to the control was taken as 100%activity in the presence of PMA-model was approximately 110%; sample containing 10 μm MANS, measurements, about 95%, gave approximately 15%reduction in EPO activity in comparison with control in the presence of PMA-reference; sample containing 50 μm MANS, measurements, approximately 78%, gave approximately 30%reduction in EPO activity in comparison with control in the Pris is under PMA-model; and the sample containing 100 μm MANS, measurements, about 65%, gave approximately 40%reduction in EPO activity in comparison with control in the presence of PMA-standard. After 2 hours, the activity of the EPO in relation to the control was taken as 100%activity in the presence of a control PMA-model was approximately 145%; the sample containing 10 μm MANS, measurements, about 130%, gave approximately 10%reduction in EPO activity in comparison with control in the presence of PMA-reference; sample containing 50 μm MANS, measurements, about 70%, gave approximately 50%reduction in EPO activity in comparison with control in the presence of PMA-reference; and a sample containing 100 μm MANS, measurements, approximately 72%, gave approximately 50%reduction in EPO activity in comparison with control in the presence of PMA-standard. Thus, after 1 and 2 hours, MANS at 50 or 100 μm significantly weakened the release of EPO. The RNS peptide had no effect on PMA-enhanced release of EPO at any time or under any of the tested concentrations. The data presented in the table below were obtained for a concentration of 50 μm of the tested peptides, which were incubated for two hours with 100 nm PMA.

Inhibition of release of lysozyme from U937 cells

Secretion of lysozyme by U937 cells were increased in PMA-stimulation, 1 h after incubation, and 2 hours after stimulation, it is peliculas even more. After 1 hour, the secretion of lysozyme by U937 cells relative to the control was taken as 100%; the secretion of lysozyme by U937 cells in the presence of a control PMA-model was approximately 210%; the sample containing 10 μm MANS, measurements, approximately 170%, gave approximately 20%reduction of lysozyme U937 cells compared with secretion in the presence of a control PMA-reference; sample containing 50 μm MANS, measurements, approximately 170%, gave approximately 20%reduction of lysozyme U937 cells compared with secretion in the presence of a control PMA-pattern; and a sample containing 100 μm MANS measurement, 115%, gave approximately 45%reduction of lysozyme U937 cells compared with secretion in the presence of a control PMA-standard. After 2 hours, the secretion of lysozyme by U937 cells relative to the control was taken as 100%; the secretion of lysozyme by U937 cells in the presence of a control PMA-model was approximately 240%; the sample containing 10 μm MANS, measurements, approximately 195%, gave approximately 20%reduction of lysozyme U937 cells compared with secretion in the presence of a control PMA-reference; sample containing 50 μm MANS, measurements, approximately 185%, gave approximately 25%reduction of lysozyme U937 cells compared with secretion in the presence of a control PMA-pattern; and a sample containing 100 μm MANS measurements, about 140%, gave approximately 40%reduction of lysozyme tile is AMI U937 compared with secretion in the presence of a control PMA-standard. Thus, the secretion of lysozyme was significantly decreased after 1 and 2 hours after stimulation under the effect of 100 μm MANS, but not under the action of 50 or 10 μm MANS. RNS peptide had no effect on PMA-enhanced secretion of lysozyme at any time or under any of the tested concentrations. The data presented in the table below were obtained for a concentration of 50 μm of the tested peptides, which were incubated for two hours with 100 nm PMA.

Inhibition of release of granzyme cells from NK-92

To assess the release of granzyme used lymphocytic cell line natural killer cells NK-92 (Gong J.H, Maki G, Klingemann H.G. Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia 8:652-658, 1994, Maki G, Klingemann H.G, Martinson J.A, Tarn Y.K. Factors regulating the cytotoxic activity of the human natural killer cell line, NK-92. J. Hematother. Stem Cell Res., 10:369-383, 2001, Takayama H, Trenn G, M.V. Sitkovsky A novel cytotoxic T lymphocyte activation assay. J. Immunol. Methods 104:183-190, 1987).

Measurement of secretion of granzyme NK-cells: level granzyme secreted by the cells of the NK-92, was measured by assessing the hydrolysis dibenzylamino ether Nα-benzyloxycarbonyl-L-lysine (BLT, EMD Bioscience, Inc.) basically, as described previously (Takayama H, Trenn G, M.V. Sitkovsky A novel cytotoxic T lymphocyte activation assay. J. Immunol. Methods 104:183-190, 1987). An aliquot of 50 μl of supernatant was transferred into a 96-well plate, and to this supernatant was added to 150 μl of 0.2 mm solution BLT and 0.22 mm DTNB (Sigma-Aldrich Co.) in Zab is perenna phosphate saline solution (PBS, pH of 7.2). The optical density at 410 nm was measured after incubation for 30 minutes at room temperature. The results were expressed as a percentage of total cellular enzyme that is present in the same number of cells lysed with Triton X-100.

As for the quantitative assessment of the level of release of granzyme cells NK-92 did not use the standard, the authors of the present invention were measured levels of release of granzyme and its intracellular levels (lysed cells), and these levels of secreted granzyme expressed as a percentage of the total level of each enzyme (level of intracellular and secreted enzyme). For level measurement of intracellular granzyme, secreted by cells, NK-92, taking appropriate aliquots of cells lysed with 0.1% Triton X-100, in order to quantify the enzyme described above. To minimize variability between cultures, all data were expressed as percentage of control. The data presented in the table below were obtained for a concentration of 50 μm of the tested peptides, which were incubated for two hours with 100 nm PMA.

Cytotoxicity

Since neither of the experiments generate toxic response in the cells was not used standard, conducted an assessment of retention/high is obozrenie LDH (data not shown) (see also, Park J-A, He F, Martin LD, Li Y, Adler KB. Human neutrophil elastase dosage hypersecretion of mucin from human bronchial epithelial cells in vitro via a PKC-δ - mediated mechanism. Am. J. Pathol. 2005, 167:651-661).

In preliminary experiments, the peptides presented in the table below, demonstrated the appropriate percentage inhibition of MPO release from human neutrophils, EPO from clone 15 cells HL-60, lysozyme from U937 cells and granzyme cells from NK-92, where MA denotes the presence myristoleic group of the substituent in the alpha N-terminal position of the peptide; Ac indicates the presence of the acetyl group of Deputy and alpha N-terminal position of the peptide; N indicates no group attached to the peptide, and NH2means the presence of the amide at the C-terminal position. The data of inhibition for several experiments were averaged. Qualitative solubility of peptides in 0,5h saline solution, pH 6.5, expressed in mg/ml, are presented below in table 3. Replacement myristoleic group other N-terminal chemical group can lead to a change in the solubility of the peptides described herein in the aquatic environment. So, for example, replacing myristoleic group on acetyl group leads to increase the solubility of these peptides in water, as shown in table 3.

Example 2

In vivosuppression induced by lipopolysaccharide (LPS) waspa the help of light under the action of the MANS peptide and related peptides

The experiment is illustrated in this example, mainly carried out by the methods described in the publications Cox, G, Crossley, J., and Xing, Z., Macrophage engulfment of apoptotic neutrophils contributes to the resolution of acute pulmonary inflammation in vivo, Am. J. Respir. Cell Mol. Biol. 12:232-237, 1995, Harada, S., Quantitative time-course profiles of bronchoalveolar lavage cells following intratracheal instillation of lipopolysaccharide in mice, Ind. Health 35:353-358, 1997; and Ulich T.R, Watson L.R, Yin SM, Guo K.Z, Wang P, Thang H, and del Castillo, J. Am. J. Pathol. 138:1485-1496, 1991.

Thus, 6-7-week-old female mice CDl weighing 15-20 grams received from the lab Charles River and maintained in cells in 5 mice per cage. Animals were given standard food for rodents and filtered waterad libitum. These animals were kept under conditions recommended by the National Institute of health (NIH), at standard temperature (64°-79°F) and a relative humidity of 30-70%.

Five experimental groups of mice, 5 animals in each group were treated with PBS, followed by treatment PBS; PBS followed by treatment of LPS; (monitorowanym) peptide MANS with subsequent treatment of the LPS; acetylated peptide SEQ ID NO:1 followed by treatment of LPS, or acetylated peptide SEQ ID NO:106, followed by LPS treatment.

Pre-treatment by intranasal instillation of peptide: a peptide according to the invention, measuredin vivoon its ability to inhibit or attenuate LPS-induced in the yesterday by drop light, was dissolved in PBS at a concentration of 1 mm. Animals shot of 0.8% isoflurane by inhalation, pre-processed shock dose of 2×10 μl of the peptide solution, intranasal introduced into one nostril for 30 minutes before subsequent instillation of LPS.

Intranasal instillation FSC: endotoxin lipopolysaccharide (LPS) (endotoxin originating fromEscherichia coliserotype 011:B4, Sigma, St Louis, MO, see .L4130, where provides information about the product Sigma under the heading "OEscherichia coli011:B4" (Lipopolysaccharides fromEscherichia coli011:B4)) was dissolved in phosphate buffered saline (PBS) at 2500 µg/ml For treatment of animals with endotoxin specified animals that were shot 0,8% isoflurane by inhalation, intranasal were administered a loading dose of 10 μl of 2500 µg/ml of endotoxin. A loading dose of 10 µl was injected into one nostril. After instillation of endotoxin, animals were monitored for the presence of shortness of breath, lethargy and reduced consumption of water/feed.

Bronchoalveolar lavage (BAL): six hours after the last instillation, the animals were anestesiologi (90 mg/kg of Nembutal) and killed by exsanguination. Light 2 times then washed with 1.0 ml-aliquot of PBS. Collected BAL fluid was centrifuged to remove cells for subsequent counting and determining leiko iternal formula. Collected lavagno liquid used for the analysis of total protein, myeloperoxidase (MPO), LDH and hemoglobin.

Analysis: Aliquots of BAL fluid was immediately used for analysis on the levels of LDH, total protein, or hemoglobin held analyzer COBAS Fara (automated analyzer COBAS FARA II, Roche Diagnostic Systems Inc., Montclair, NJ). An aliquot of BAL fluid was frozen at -80°C for subsequent quantification of myeloperoxidase (MPO)conducted using mouse-specific ELISA analysis (Cell Sciences, Inc., Canton, Mass). BAL data were analyzed by standard methods to assess differences between control and treatment groups. The results showing inhibition or attenuation of inflammation under the influence of the tested peptide presented in the following tables.

Table 4
Average values of inflammatory markers in the presence of the MANS peptide, MA-GAQFSKTAAKGEAAAERPGEAAVA, SEQ ID NO:1
The processing circuitThe total number of counted cellsThe total number of counted neutrophils% of neutrophils from the total number of cellsMPO (ng/ml)Total protein (µg/ml)LDH (units/l) Hb (g/DL)
PBS/PBS (n=51570202931718,73,28125,6068,200,00
PBS/LPS (n=526420011006141,728,98272,4060,400,19
MANS/FSC n=72084576448130,99,49175,0068,570,05

Table 5
Average values of inflammatory markers in the presence of N-terminal acetylated analogue of the MANS peptide, Ac-GAQFSKT AAKGEAAAERPGEAAVA, SEQ ID NO:1
The processing circuitThe total number of counted cellsThe total number of counted neutrophils% of neutrophils from the total number of cellsMPO (ng/ml)Total protein (µg/ml) LDH (units/l)Hb (g/DL)
PBS/PBS (n=5894401977022,1the 5.45230,684,00,00
PBS/LPS (n=525136016457865,537,90153,4to 89.90,01
AC-SEQ ID No. 1/LPS (n=525440010549941,4730,79182,7574,50,01

Table 6
Average values of inflammatory markers in the presence of acetylated peptide Ac-GAQFSKTAAK, SEQ ID NO:106
The processing circuitThe total number of counted cellsThe total number of counted neutrophils% of neutrophils from the total number of cellsMPO (ng/ml) Total protein (µg/ml)LDH (units/l)Hb (g/DL)
PBS/PBS (n=53126206652121,34,88113,861,800,00
PBS/LPS (n=53276808007724,47,19116,478,200,00
AC-SEQ ID No. 106/LPS (n=530568891703,01,50131,0106,860,00

Table 7
These inhibition of markers of inflammation under the action of the MANS peptide (Myr-SEQ ID NO:1), the tested peptides (Ac-GAQFSKTAAKGEAAAERPGEAAVA), SEQ ID NO:1 and Ac-GAQFSKTAAK, SEQ ID NO: 106, compared with the data obtained in PBS/LPS-processing
The processing circuitInhibition of neutrophil migrationInhibition of MPO
MANS/LPS41,4%67,2%
SEQ ID NO:1/LPS35,9%18,75%
AC-SEQ ID NO:106/LPS88,5%79,1%

PBS/PBS means of the introduction of only PBS as a control without the addition of the endotoxin LPS to stimulate chemotactic migration of neutrophils; PBS/LPS means adding LPS (endotoxin) to stimulate chemotactic migration of neutrophils; MANS/FSC pre-processing means of the MANS peptide in PBS, followed LPS-stimulation to induce migration of neutrophils. The percentage of neutrophils of total number of cells in groups of LPS treatment decreased from 41.7 per cent to 30.9% in the processing of the MANS peptide; from 65.5% to 41,47% in the processing of peptide Ac-GAQFSKTAAKGEAAAERPGEAAVA, SEQ ID NO:1, and from 24.4 percent to 3.0 percent in the processing of peptide Ac-GAQFSKTAAK, SEQ ID NO:106. The measured levels of MPO in groups of LPS treatment decreased from 28,98 ng/ml to 9,49 ng/ml in the processing of the MANS peptide, or 37.9 ng/ml to 30,79 ng/ml in the processing of the acetylated peptide with SEQ ID NO:1 and from 7,19 ng/ml to 1.50 ng/ml in the processing of the acetylated peptide with SEQ ID NO:106.

Example 3

Mouse model of COPD induced by ozone

Oxidative stress caused by chemical irritants, such as oz is h, is a well-known feature of chronic obstructive pulmonary disease (COPD). Cm. publication Repine JE, Bast A, Lankhorst I, and the Oxidative Stress Study Group, Am. J. Respir. Crit. Care Med. 156:341-357, 1997, and Harkema J.R and Hotchkiss J.A, Toxicology Letters, 68:251-263, 1993.

10-week-old female Balb/C mice were obtained from the laboratory of Charles River and maintained in cells in 5 mice per cage under conditions recommended by the National Institute of health (NIH). Animals were given standard food for rodents and filtered waterad libitum. Mice in the three treatment groups containing 5 animals each, were anestesiologi by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (20 mg/kg), and then pre-treated by intratracheal injection of 25 μl of the same PBS, or 1.0 mm solution of MANS peptide in PBS, or a solution of 1.0 mm slice acetylated peptide MANS Ac-GAQFSKTAAK called here acetylated SEQ ID NO:106 in PBS. After 30 minutes, the animals were placed in the appropriate specially appointed chamber for supplying ozone and the charge air. Animals were treated with ozone for 2 hours (at ozone concentrations of 1-10 ppm) slightly modified method described by Haddad et al., 1995 (Haddad E-B, Salmon M, Sun J, Liu S, Das A, Adcock I, Barnes P.J., Chung KF, FEBS Letters, 363:285-288, 1995). Ozone was generated using an ozone generator model OL80F/B, OzoneLab, Burton, British Columbia, Canada. Subsequent continuous monitoring is ing ozone concentration was performed using the analyzer Teledyne Photometric 03 (model 400E, Teledyne Instruments, City of Industry, CA). Mice of two additional groups, which have not been subjected to any treatment, were treated with ozone in the same conditions, or the pressurized air in conditions analogous to treatment with ozone, but in the absence of ozone. After treatment, animals were killed by exsanguination, and light 2 times then washed with 1.0 ml-aliquot of PBS. Collected bronchoalveolar lavage (BAL) was centrifuged to remove cells for subsequent counting and determination of the leukocyte formula. Collected lavagno liquid used for the analysis of protein and for additional analysis on IL-6, IFNγ and KC (murine analog of IL-8)by using ELISA (using analytical kits obtained from R&D Systems, Minneapolis, MN).

The percentage of inhibition of migration of neutrophils in BAL fluid treatment groups with respect to the data obtained for the control group treated with only PBS, presented in the table below.

MANS + ozone
Table 8
The inhibition induced by ozone migration of neutrophils MANS peptide and a peptide with an acetylated SEQ ID NO:106, Ac-GAQFSKTAAK
Processing group% inhibition of migration of neutrophils in BAL-fluid
93,0
AC-SEQ ID NO:106 + ozone81,2
PBS + ozoneunacceptable
Only the pressurized airunacceptable

The concentration of IL-6 in PG/ml in BAL fluid depending on prior intratracheal injection and subsequent treatment with ozone was prepared as follows. We have received the following levels of IL-6: approximately 364,5 PG/ml in the group of mice pretreated with peptide MANS, and then the ozone; approximately 130,4 PG/ml in the group of mice pre-treated acetylated fragment of the MANS peptide, Ac-GAQFSKTAAK (SEQ ID NO:106), and then ozone; approximately 1041,3 PG/ml in the group of mice pre-treated PBS and ozone, about to 43.2 PG/ml in the group of mice injected directly processed air without any pre-processing.

The concentration of KC in PG/ml in BAL fluid depending on prior intratracheal injection and subsequent treatment with ozone was prepared as follows. We have received the following levels KS: approximately 183,6 PG/ml in the group of mice pretreated with peptide MANS, and then the ozone; approximately 159,7 PG/ml in the group of mice has preliminarily is treated acetylated fragment of the MANS peptide, Ac-GAQFSKTAAK (SEQ ID NO:106), and then ozone; approximately 466,6 PG/ml in the group of mice pre-treated PBS and ozone, approximately 36,3 PG/ml in the group of mice injected directly processed air without any pre-processing.

The concentration of IFNγ in PG/ml in BAL fluid depending on prior intratracheal injection and subsequent treatment with ozone was prepared as follows. We have received the following levels of IFNγ: approximately 7,4 PG/ml in the group of mice pretreated with peptide MANS, and then the ozone; approximately 3.6 PG/ml in the group of mice pre-treated acetylated fragment of the MANS peptide, Ac-GAQFSKTAAK (SEQ ID NO:106), and then ozone; approximately 8,6 PG/ml in the group of mice pre-treated PBS and ozone, approximately 5,0 PG/ml in the group of mice injected directly processed air.

Introduction ozone mice resulted in a significant increase in the number of infiltrated neutrophils and the levels of IL-6 and KC at BAL. Compared with the control group of mice that were previously treated with PBS, each mouse in groups pretreated with MANS peptide, acetylated peptide Ac-GAQFSKTAAK, i.e. acetylated SEQ ID NO:106, was found to decrease in the infiltration of neutrophils in the BAL fluid after treatment with ozone (for example, 93% ± 10%and 81% ± 10%, respectively, compared to PBS control). In parallel, the MANS peptide and acetylated peptide, namely acetylated SEQ ID NO:106 after treatment with ozone is also markedly reduce the concentration of KC (for example, 65,8% ± 10% and 71.3% ± 10%, respectively, compared to PBS control), and the levels of IL-6 (e.g., 67,8% ± 15%, MANS and 91.3% ± 15%, acetylated SEQ ID NO:106, compared to PBS control), but have little impact on the levels of interferon-γ. Overall, these data showed that the MANS peptide and acetylated peptide SEQ ID NO:106 significantly reduce or inhibit induced ozone migration of neutrophils into the Airways, and selectively reduce the levels of chemokines and cytokines. The levels of IL-6 in BAL fluids obtained from animals pretreated with MANS peptide or acetylated peptide SEQ ID NO:106, inhibited by approximately 68% and 91%, respectively, compared with levels in animals pre-treated PBS. In addition, the levels of KC at BAL fluids obtained from animals pretreated with MANS peptide or acetylated peptide SEQ ID NO:106, inhibited by approximately 65% and 71%, respectively, compared with levels in animals pre-treated PBS.

Example 4

The model of chronic bronchitis

This procedure, described in the publications Voynow J.A, Fischer B.M, Malarkey D.E, Burch LH, Wong T, Longphre M, Ho S.B, Foster W., Neutrophil Elastase dosage mucus cell metaplasia in mouse lung, Am. J. Physiol. Lung Cell Mol. Physiol. 287:L1293-L1302, 2004, was conducted to build a model of chronic bronchitis in mice. In particular, hyperplasia of goblet cells, which is the hallmark of chronic bronchitis, induced by permanent treatment of mice with human elastases neutrophils (NE)introduced into the respiratory tract via instillation.

Human NE intratrahealno was aspirated in male Balb/c mice. Just purchased 30 mice (weighing 25-30 g) from a vendor, such as Jackson Laboratories, Bar Harbor, ME. Mice were kept in a 12-hour cycle of day and night with free access to feed and water. Animals were injected NE by oropharyngeal aspiration on days 1, 4 and 7. Immediately after anesthesia by inhalation isoflurane (IsoFlo from Abbott Laboratories) and systems of anesthesia gas on an open path from Stoelting, animals were hung by their upper incisors (front teeth) on an inclined plane at an angle of 60°, and the volume of liquid containing human NE [50 µg (43,75 units)/40 ál of PBS (Elastin Products, Owensville, MO)], was introduced by applied on the elongated tongue of the animal, extending to the distal of the oropharynx. When extended the language of the animal cannot swallow the liquid, and therefore, the amount of liquid gets into the respiratory tract.

7 days after the last NE-processing, if hyperplasia Bo is lovenich cells, modeled in Airways in chronic bronchitis, reached its maximum (see Voynow et al, 2004), mice (5 animals per group) by intratracheal instillation was administered 50 μl of either PBS (as control)or 100 μm solution of peptide MANS solution, RNS peptide or peptide solution, such as acetylated peptide SEQ ID NO:106, dissolved in PBS. After 15 minutes, mucus secretion stimulated by introduction of methacholine using aerosol inhalers Buxco, resulting received a fine aerosol of methacholine coming at a concentration of approximately 60 mm for 3 minutes. 15 minutes after the introduction of metacholine, mice were killed by inhalation of 100% gas CO2.

Histochemical analysis

After the processing described above, the lungs of the animals were purged to remove blood, and then breathed Wednesday OCT (Wednesday optimally with low temperature) (Sakura Finetck, Torrance, CA), half diluted in physiological solution. The lungs were immersed overnight in 10% formaldehyde in PBS at 4°C and embedded in paraffin. 5 µm sections were treated with periodic acid-Schiff/hematoxylin dye for the mucines respiratory tract, for example, as described in publications Singer M, B.B. Vargaftig, L.D. Martin, J.J. Park, A.D. Gruber, Li Y, K.B. Adler, A MARCKS-related peptide blocks mucus hypersecretion in a murine model of asthma., Nature Medicine 10:193-196, 2004.

Histology the economic index mucus

Histological index of mucus (Whittaker L, Niu N, Temann U-A, Sloddard A. Flavell R.A, Ray A, Homer R.J, and Cohn L, Interleukin-13 mediates a fundamental pathway for airway epithelial mucus induced by CD4 T cells and interleukin-9, Am. J. Respir. Cell Mol. Biol. 27:593-602, 2002) was determined by AB/PAS-stained sections, which cover the Central and peripheral Airways. Slides were observed through a 10X lens and the image was shot on digital videocamera. Each animal received a minimum of four representative snapshot of the transverse or sagittal slices of the respiratory tract. Visualize you only the respiratory tract, which had full circle, and the image of these slices were analyzed. The respiratory tract that are opened directly in the alveolar space, not analyzed. The degree of PAS-positive staining in each image of the respiratory tract was estimated by semi-quantitative researcher who did not know about the conditions of processing of each slice, in accordance with the following 5-point scoring system: score 0, PAS staining is absent; grade 1, PAS-staining was observed in 25% or less of the epithelium of the respiratory tract; score 2, PAS-staining was observed in 26-50% of the epithelium of the respiratory tract; score 3, PAS-staining was observed in 51-75% of the epithelium of the respiratory tract; score 4, PAS staining was observed on >75% of the respiratory epithelium ways. This rating system IP is alzueta to calculate the index of mucus in each group, and the results of this assessment are presented as mean ± srcpos.

All results are presented as mean ± srcpos (n=5 animals, 10-20 slices for each animal). The significance levels were calculated using one-way analysis of variance ANOVA, and then using the criterion Scheff using the computer program SPSS 6.1 (* = significance of the data compared with a threshold value of p<0,05).

Example 5

In vivotests

The purpose of the following series of experiments is to determine the influence of the peptides according to the invention after their deliveryin vivoor by local instillation to the site of inflammation, or intravenous injection on the development of inflammation compared with control peptides, such as RNS. For this purpose used two models: (i) a murine model of inflammation of the air pocket and (ii) a murine model of peritonitis-induced thioglycolate. Both of these models are well characterized model of inflammation in the development of which the important role played by neutrophils. Model air pocket allows you to determine the influence of peptides on a rapid course of inflammation (approximately 4 hours), and the model of peritonitis can be used to estimate the effect of peptides for a longer period of inflammation (approximately 24 hours).

General Protocol e is speriment

In order to determine the effect of the peptides described in the present application, for each of the two models, namely models i.v. delivery of peptides and model local delivery of peptides, conducted four studies. In each study, there were 2 experimental groups, namely control group without inflammation (processed by the media) and the group with induced inflammation (i.e. treated stimulator of inflammation). Each group was divided into 5 and optional 6 subgroup treatment n=6 for each subgroup. These subgroups have been processed, for example a carrier, MANS, RNS, test peptide, optionally a peptide having a "scrambled" sequence of test peptide, where this "scrambled" sequence is a two-part sequence, "peptide-SCR", and dexamethasone. Dexamethasone is used as the reference anti-inflammatory drugs. The selection of appropriate doses for i.v. injection or local instillation was performed on the basis of preliminary experiments to determine the dependence "dose-response". Experimental doses that are defined on the basis of inhibitory activity MANS in human neutrophils, up to 1 mg/kg i.v. one-time delivery or the final concentration of 50 μm, administered locally (in air pocket or I.P. Pavlova.). Dose for i.v. D. the rate was selected based on the volume of distribution of 2 l/kg

Model of inflammation of the air pocket:

Tests for infiltration of neutrophils and inflammation in the murine air pocket was carried out as described in the publication C.B. Clish, O'brien J.A., Gronert K., Stahl, G.L., N.A. Petasis, Serhan C.N. Local and systemic delivery of a stable aspirin-triggered lipoxin prevents neutrophil recruitmentin vivo. Proc. Natl. Acad. Sci USA. 1999 Jul 6; 96(14):8247-52. Thus, male white mice BALB/c (6-8 weeks) were anestesiologi isofluorane, and the volume of the dorsal air pockets increased by subcutaneous injection of 3 ml of sterile air on day 0 and 3. On day 6 the mice were anestesiologi with isoflurane, and then threw in the media, MANS, RNS, test peptide or, but not necessarily, the peptide-SCR, or by i.v. injection loading dose into the tail vein in 100 ál of sterile 0,9% saline solution, or topically air pocket in 900 μl of PBS-/-(phosphate buffered saline, Dulbecco, not containing magnesium ions or calcium, BioWhittaker). Dexamethasone (Sigma), enter or i.v. at a dose of 0.1 mg/kg in 100 μl sterile 0,9% saline solution, or topically at a dose of 10 mg in 900 μl of PBS-/-serves as a reference anti-inflammatory drugs. Inflammation in the air pocket induced by local injection of recombinant murine tumor necrosis factor α (TNF-α, 20 ng) (Boehringer Mannheim)dissolved in 100 µl of sterile PBS. 4 hours after the first iny the functions of TNF-α, air pockets mice, shot by isofluorane, twice washed with 3 ml of sterile PBS. The aspirates were centrifuged at 2000 rpm for 15 minutes at 23°C. Supernatant was removed, and cells suspended in 500 ál PBS. Aliquots of the supernatant were analyzed for concentrations of inflammatory mediators (optional, except TNFα), MPO activity and perechislenie lipids.

The total number of cells in the cell suspension was counted under an optical microscope using hemocytometer. Resuspendable cells aspirate (50 µl) on slides microscope was added to 150 μl of 30% BSA and centrifuged at 2200 rpm for 4 minutes on cytocentrifuge. The leukocyte formula cells, centrifuged cytocentrifuge and stained with Wright-Giemsa, used to count the absolute number of cells of each type to bleed and have exudate air pockets. For analysis under a microscope, the tissue thickness 6 mm obtained by punch biopsy tissue (Acu-Punch, Acuderm)were fixed in 10% buffered formaldehyde. Then the samples were embedded in paraffin, and prepared sections, and these sections were stained with hematoxylin-eosin. The number of neutrophils in histological sections was determined by counting the number of cells/hpf (in the field of view at high magnification microscope). As a control for derma raw is on the air pocket was the distal region of the dermis.

Data were presented as the total number of neutrophils, monocytes, eosinophils, basophils and lymphocytes to bleed and have exudate or the number of neutrophils in the tissue in the field of view at high magnification microscope. The values obtained were expressed as mean ± srcpos. (n=6). The significance of the effect of any treatment on the migration was determined using ANOVA. The value of P<0,05 was considered significant.

Example 6

Model inflamed peritoneum

Male BALB/c mice (6-8 weeks) were used to create the model induced thioglycollate peritonitis, as described in the publication Tedder T.F, Steeber D.A, Pizcueta p L-selectin-deficient mice have impaired leukocyte recruitment into inflammatory sites. J. Exp. Med. 1995 Jun 1, 181(6):2259-64. Media, MANS, RNS, test peptide and, optionally, the peptide-SCR was introduced by the injection of a test dose of either the tail vein in 100 ál of sterile 0,9% saline solution, or topically in the peritoneum in 900 μl of PBS-/-immediately prior to I.P. Pavlova.-injection thioglycolate. Dexamethasone, administered or i.v. at a dose of 0.1 mg/kg in 100 μl sterile 0,9% saline solution, or topically at a dose of 10 mg in 900 μl of PBS-/-serves as a reference anti-inflammatory drugs. Inflammation was induced by intraperitoneal injection to mice 1 ml of thioglycolate (3% wt./about, Sigma Immunochemicals). 24 hours after induction of inflammation, mice were subjected to humane avtan the Ziya and peritoneum were injected with 5 ml warm environment (environment at ~37°C (RPMI 1640, 2% FCS, and 2 mm EDTA), and then did a light massage of the abdominal cavity. The aspirates lavagno fluid in the abdominal cavity was centrifuged at 2000 rpm for 15 minutes at 23°C. Supernatant was removed, and cells suspended in 500 ál PBS. Aliquots of the supernatant were analyzed for MPO activity, concentrations of inflammatory mediators and perechislenie lipids.

The total number of cells in the cell suspension was counted under an optical microscope using hemocytometer. Resuspendable cells aspirate (50 µl) on slides microscope was added to 150 μl of 30% BSA and centrifuged at 2200 rpm for 4 minutes on cytocentrifuge. The leukocyte formula cells, centrifuged cytocentrifuge and stained with Wright-Giemsa, used to count the absolute number of cells of each type to aspirate air pocket.

Data were presented as the total number of neutrophils, monocytes, eosinophils, basophils and lymphocytes to bleed and have exudate. The values obtained were expressed as mean ± srcpos. (n=6). The significance of the effect of any treatment on the migration was determined using ANOVA. The value of P<0,05 was considered significant.

Degranulation

As a marker of degranulation used myeloperoxidase. Myeloperoxidase activity in the cell supernatant obtained from lavagno liquid air is nogo pocket or peritoneum, evaluated and analyzed as described above using the TMB method.

Concentrations of inflammatory mediators

Concentrations of key proinflammatory mediators TNF-α, IL-1β, IL-10, IL-6, KC and PGE2 in lavagno liquid air pocket, and the peritoneum was determined using commercially available ELISA kits (R&D Systems) according to manufacturer's instructions.

Perechislenie lipids

The concentration of F2-isoprostanes is a sensitive and specific indicator of oxidative damage caused by the release of intermediates reactive oxygen species from neutrophils and other cells (Milne G.L, Musiek signals of E.S., Morrow J.D. F2-isoprostanes as markers of oxidative stressin vivo: an overview. Biomarkers are. 2005 Nov; 10 Suppl 1:S10-23). The concentration of F2-isoprostanes was determined in supernatant exudate air pockets and peritoneum using a commercially available ELISA-analysis (8-Isoprostane EIA, Cayman Chemical) according to manufacturer's instructions.

Results

The experiment was considered successful if local or systemic delivery of the tested peptides by one or more of the above described methods of inhibiting the release of inflammatory mediators, leading to attenuation of inflammation.

Active fragments of the peptides according to the invention inhibit the influx of neutrophils in the inflamed air pocket sludge is in the inflamed peritoneum and inhibit the degranulation of neutrophils in these organs, which leads to decreased activity of MPO, perechisleniya lipids and production of inflammatory mediators.

The above examples are only for illustrative purposes and should not be construed as limiting the scope of the invention. Scope of the present invention is defined by the following claims, which includes described here are equivalent.

1. The method of inhibition of MARCKS-associated release of at least one mediator of inflammation of granules of at least one inflammatory cells in the tissue and/or fluid of the individual, including:
introduction to the specified fabric and/or liquid therapeutically effective amount of a pharmaceutical composition containing at least one peptide acetylated at N-terminal alpha-amino group, with an amino acid sequence selected from the group consisting of:
(a) sequence of 24 amino acids, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1);
(b) amino acid sequence of size 4, but not more than 23, consecutive amino acids of the reference sequence GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1);
(c) sequences with the same amino acid substitution, which differ by one amino acid from the sequence defined in subparagraph (b), where a single substitution selected from the group consisting of G in position 1 And replaced, And in position 2 the Deputy is Yong K, Q in position 3 is replaced by a or E, F in position 4 is replaced by A, S at position 5 is replaced And, in the 6 position is replaced And, T in position 7 And replaced, And in position 8 replaced with K, K in position 10 replaced with A, G at position 11 is replaced by a and E at position 12 is replaced with A; and
(d) sequence with two amino acid substitutions that differ by two amino acids from the sequence defined in subparagraph (b), where two substitutions selected from the group consisting of And in positions 2 and 8 replaced It, and in positions 6 and 10 And replaced;
where C-terminal amino acid of the peptide with the amino acid sequence defined in subparagraph (a)or (b)or (C)or (d), chemically modified, for example, amidon ammonia or remodification on the group of carboxylic acid-end; and
where the specified peptide optionally combined with a pharmaceutically acceptable carrier and in an amount therapeutically effective to attenuate release of mediators of inflammation, reduces the release of mediators of inflammation from at least one inflammatory cells compared with the release rate of the specified mediator of inflammation of at least one inflammatory cells of the same type, in the absence of the indicated peptide.

2. The method according to claim 1, where the specified peptide acetylated at N-terminal alpha-amino group selected from the group consisting of acetyl-GAFSKTAAK (SEQ ID NO:106), acetyl-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1), acetyl-GAQFSKTAAKGEAAAERPGE (SEQ ID NO:11), acetyl-GAQFSKTAAKGEAAAE (SEQ ID NO:37), acetyl-AKGEAAAERPGEAAVA (SEQ ID NO:45), acetyl-GAQFSKTAAKGE (SEQ ID NO:79), acetyl-AAAERPGEAAVA (SEQ ID NO:91), acetyl-GAQFSKTAA (SEQ ID NO:121), acetyl-TAAKGEAA (SEQ ID NO:143), acetyl-RPGEAAVA (SEQ ID NO:153), acetyl-AAKGEA (SEQ ID NO:179), acetyl-AKGE (SEQ ID NO:219), acetyl-GAQFSKTAAKGE-NH2(SEQ ID NO:79), acetyl-AQFSKTAAKGE-NH2(SEQ ID NO:93), acetyl-QFSKTAAKGE-NH2(SEQ ID NO:108), acetyl-FSKTAAKGE-NH2(SEQ ID NO:124), acetyl-SKTAAKGE-NH2(SEQ ID NO:141), acetyl-KTAAKGE-NH2(SEQ ID NO:159) and acetyl-AKGE-NH2(SEQ ID NO:219).

3. The method according to claim 1 or 2, where the specified peptide consists of at least ten contiguous amino acid residues.

4. The method according to claim 3, where the specified peptide consists of acetyl-peptide 106 (SEQ ID NO:106).

5. The method according to claim 1 or 2, where the amino acid sequence of the peptide includes a contiguous amino acid residues AKGE.

6. The method according to claim 1, where the specified peptide consists of at least six contiguous amino acid residues.

7. The method according to claim 1 or 2, where the specified peptide amitirova ammonia at the C-terminal alpha-amino acids.

8. The method according to claim 1 or 6, where the specified peptide includes the amino acid sequence of (b) and does not begin with N-terminal amino acids of the reference sequence GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1).

9. The method of claim 8, where the specified peptide amitirova ammonia at the C-terminal alpha-amino acids.

10. The method according to claim 1, where aminos the PCI-e slot sequence with a single substitution (s) selected from the group consisting of GKQFSKTAAKGE (SEQ ID NO:233), GAQFSKTKAKGE (SEQ ID NO:234), GAQASKTAAK (SEQ ID NO:236), GAQASKTAAKGE (SEQ ID NO:237), GAEFSKTAAKGE (SEQ ID NO:238), GAQFSKTAAAGE (SEQ ID NO:239), GAQFSKTAAKAE (SEQ ID NO:240), GAQFSKTAAKGA (SEQ ID NO:241), AAQFSKTAAK (SEQ ID NO:242), GAAFSKTAAK (SEQ ID NO:243), GAQFAKTAAK (SEQ ID NO:244), GAQFSATAAK (SEQ ID NO:245), GAQFSKAAAK (SEQ ID NO:247), GAQFSKTAAA (SEQ ID NO:248), GAQASKTA (SEQ ID NO:250) and AAGE (SEQ ID NO:251).

11. The method according to claim 1, where the amino acid sequence with two substitutions selected from the group consisting of GKQFSKTKAKGE (SEQ ID NO:235) and GAQFSATAAA (SEQ ID NO:249).

12. The method according to claim 10 or 11, where the specified peptide amitirova ammonia on the C-terminal alpha-amino acid.

13. The method according to claim 1, where the specified inflammatory cell selected from the group consisting of a leukocyte, granulocyte, neutrophil, basophil, eosinophil, monocyte, macrophage, and their combinations.

14. The method according to claim 1 or 13, where the specified mediator of inflammation is selected from the group consisting of myeloperoxidase (MPO), peroxidase eosinophils (EPO), the main major protein [ICBMs], lysozyme, granzyme, histamine, proteoglycan, proteases, chemotactic factor, a cytokine, a metabolite of arachidonic acid, defensin, protein, increasing permeability of the bacteria (BPI), elastase, cathepsin G, cathepsin B, cathepsin D, beta-D-glucuronidase, alpha-mannosidase, phospholipase A2, chondroitin-4-sulfate, proteinase 3, lactoferrin, collagenase, activator of complement, complement receptor, receptor N-formylmethyl-leucyl-vanilla the ina (FMLP), the laminin receptor, cytochrome b558, macrophage chemotactic factor, histaminase, protein, bind with vitamin B12, gelatinase, plasminogen activator, beta-D-glucuronidase and their combinations.

15. The method according to claim 1, where the specified effective amount of the indicated peptide, reduces the level of release of mediators of inflammation, include the amount of a peptide inhibiting the degranulation, which reduces the level of a mediator of inflammation released from at least one inflammatory cells, (i) from 1% to about 99%, or (ii) from 5-50% to 99% compared to the level emitted from at least one inflammatory cell in the absence of the indicated peptide.

16. The method according to claim 1, where the specified mediator of inflammation associated with respiratory disease.

17. The method according to clause 16, where the specified respiratory disease selected from the group consisting of asthma, chronic bronchitis, COPD and cystic fibrosis.

18. The method according to claim 1, where the aforementioned introduction selected from the group consisting of local administration, parenteral administration, rectal administration, pulmonary administration, intranazalnogo injection and oral administration.

19. The method according to p, where the specified pulmonary introduction includes the introduction of spray.

20. The method according to claim 19, where the specified aerosol obtained from the inhaler dry powder is CA, nebulizer with a metering valve or a nebulizer.

21. The method according to claim 1, further comprising an introduction to the specified individual of the second molecule selected from the group consisting of antibiotics, antiviral compounds, antiparasitic compounds, anti-inflammatory compounds and immunomodulator.

22. The method according to claim 1, where the specified mediator of inflammation associated with a disease selected from the group consisting of intestinal diseases, skin diseases, autoimmune diseases, pain syndrome, and combinations thereof.

23. The method according to item 22, where the specified intestinal disease selected from the group consisting of ulcerative colitis, Crohn's disease and irritable bowel syndrome.

24. The method according to item 22, where the specified skin disease selected from the group consisting of rosacea, eczema, psoriasis and acne severe.

25. The method according to claim 1, where the specified mediator of inflammation associated with arthritis.



 

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FIELD: chemistry.

SUBSTANCE: invention relates to molecular pharmacology and specifically to a peptide which is part of an interleukine-15 (IL-15) sequence which can inhibit biological activity of the said molecule.

EFFECT: obtaining a peptide which inhibits T cell proliferation induced by IL-15, and apoptosis caused by tumour necrosis factor when bonding with the alpha subunit of the (IL-15R) receptor.

8 cl, 4 dwg, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to application of peptide of formula (I): ((X)1(Y)m)n where peptide contains 3 to 200 amino acids and where 1, m and n represent integers within 0 to 10; X and Y which can be identical or different, represent cationic amino acids chosen from arginine and lysine in preparing drugs for treating fungal infection.

EFFECT: ensured applicability of peptide for preparing a drug for treating fungal infection.

26 cl, 53 dwg, 2 tbl, 19 ex

Rgd-like peptides // 2396271

FIELD: chemistry.

SUBSTANCE: invention discloses novel synthetic RGD-like peptides capable of dose-dependant inhibition of thrombocyte aggregation.

EFFECT: obtaining novel compounds capable of dose-dependant inhibition of thrombocyte aggregation.

2 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and is a peptide which induces killer T cells ex vivo and which has an amino acid sequence as shown in one SEQ ID NOS: from 1 to 3. The disclosed peptide is used in an ex vivo agent for inducing anti-tumour immunity, in an ex vivo agent for inducing antigen-presenting cells, in an ex vivo agent which induces tumour-reactive T cells, as well as in an ex vivo pharmaceutical agent when treating or preventing tumours. The invention also relates to an antibody against the said peptide.

EFFECT: disclosed agents enable identification of glypican-3-derivative peptide, which can bond with HLA-A2, and activation of human killer T cells in order to provide an immunotherapy agent which may be effective in approximately 40% Japanese patients suffering from certain types of malignant tumours, accompanied by high level of GPC3 expression.

7 cl, 4 dwg, 1 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to contrast agents which contain a peptide vector linked with uPAR, marked with a visualising group.

EFFECT: obtaining a contrast agent for detecting urokinase plasminogen activator receptor.

6 cl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to the application of a biologically active peptide which represents the amino acid sequence SEQ ID No.1.

EFFECT: preparation of a drug for modulation of at least one of the following conditions: fatigue, liver glycogen level and blood lactic acid level.

30 tbl, 14 ex

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