Cytomegalia lipophilic peptides for the modulation of the activity of the immune system and suppress inflammation

 

The invention relates to oligopeptides comprising the amino acid sequence of the B-X-X-X-B-X-X-X-J-Tyr, which is Lys or Arg; X represents any amino acid, other than charged aliphatic amino acid or its D-isomer; and J is Gly, Lys or Arg; these amino acids are found in nature L-isomer, D-isomer and norleucine, and where specified Oligopeptide includes other than (a) naturally occurring sequencedomain antigen In human leukocyte (HLA-B) 75-84, (b) a naturally occurring sequence of the transmembrane sequence-chain T-cell receptor of human rights and (in) consistency, which is the mutant with respect to either (a) or (b) having no more than two mutations as an active part of the above oligopeptides; the method of suppressing the activation limfozitah cells, the method of transplantation of donor organs or mammalian cells, methods of inhibiting protein synthesis of inflammatory cytokines by cells capable of producing the specified protein inflammatory cytokine, the method of suppressing an inflammatory response in a mammal, the method of modulation of the activity hem-comprising of EN zymes-break-before:always;">

The technical FIELD of the INVENTION This invention relates to the field of chemistry and medicine, more specifically to novel peptides useful for the modulation of the activity of immune system cells and to suppress inflammation.

The prior art, the Immune system is an extremely complex combination of cells and compositions, which protects the owner, representing a mammal, from a wide variety of pathogens, and at the same time carries out the control in his body for harmful aberrations, such as neoplasms. One branch of the immune system includes cells that carry out functions of the immune system, the lymphocytes, such as originating from bone marrow b-lymphocytes and originating from the thymus gland cells T-lymphocytes and cells are natural killer cells (NK), and (b) mononuclear phagocytes, including monocytes and macrophages. At that time, as lymphocytes are primarily associated with specific immune responses due to their ability to specifically recognize and distinguish antigenic determinants, mononuclear phagocytes often carry out the total destruction of alien microorganisms by phagocytosis, as well as the synthesis and sydnye antigen to T-lymphocytes. Function limfozitah cells and mononuclear phagocytes are closely related and are important for the proper functioning of the immune system.

One of the important subpopulations limfozitah cells are T-lymphocytes, which got its name due to the fact that they are produced by the thymus (thymus gland). T-lymphocytes are a group of cells, including the different cells, which can be cytotoxic, with numerous mechanisms that cause cell death, or activating, by the synthesis of various cytokines, which function is to activate other cells. Cytotoxic T lymphocytes (CTLs) are being limited to a specific antigen major histocompatibility complex (MHC), and Express T-cell receptor on the cell surface that includes bothandcircuit, and which has a specific affinity to a specific complex of MHC-related peptide in the groove of this MHC. CTLs automatically adjusted so that normally they do not act against those cells which are in the groove of the peptide is endogenous to the host. However, in the case where the MHC is the destroy them. Other limfocitna cells, which play an important role in the immune response, include b-lymphocytes and NK-cells (natural killer cells), and the activity of both of these cell types may be affected by other cells of the immune system and various polypeptides cytokines.

Mononuclear phagocytes are the other main population of cells of the immune system and consist of cells having a common line of differentiation, the main function is phagocytosis. Mononuclear phagocytes originate from precursor cells of the trunk bone marrow, and after maturation and subsequent activation they can acquire various morphological forms, including incompletely differentiated monocyte cells and macrophages. Proper functioning of mononuclear phagocytes depends on ability how to produce different cytokine proteins, and to respond to them.

Cytokines, such as various interferons, interleukins, tumor necrosis, chemokines, hematopoietic growth factors and factors of inhibition of cell migration are a group of different proteins synthesized by a wide variety of different types of cells of the immune system. The most important is that cytokines are produced by the main cytokines produced during the effector phase as a natural, so specific immunity and serve as mediators and regulators of both immune and inflammatory reactions. Cytokines and other polypeptide hormones, begin their action by binding to specific receptors on the surface of target cells, and their activation often leads to inflammatory reactions.

Although activation of the immune response and induced cytokine inflammatory response is extremely important for the health of the host and for the proper functioning of the immune system, there are several situations in which this activation is undesirable. One of these specific situations associated with transplantation, which very rarely identical compatibility between the MHC antigens of the donor and recipient. The second situation is related to the fact that the "failure" part of CTLs they attack cells that have both MHC and related peptides are endogenous, that occurs in autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM). In addition, there are cases when mediated by cytokines functions of the inflammatory response adversely affect the health of the host, for example, inflammatory reactions associated with diseases such as septicemia activation of CTLs unwanted, became immunosuppression. However, immunosuppressants, such as cyclosporine A, FK-506, and the like, have numerous unwanted side effects. In addition, different approaches have been used, aimed at controlling or suppressing inflammatory reactions, however, many of these approaches also have one or more undesirable effects. There is therefore considerable interest in the identification of new agents that can inhibit activation limfozitah cells, in particular CTLs, and at the same time have less universal immunosuppressive effects on the immune system and have fewer side effects, to leave the owner with a substantial part of its immune system to protect against accidental infections. There is also considerable interest in the identification of new agents, the function of which is to control or suppression of inflammatory reactions.

In the last few years there have been reports that the oligopeptides effective to modulate the activity of the immune system and increasing the life expectancy of allogeneic grafts. These oligopeptides based on1domain antigen In human leukocyte (HLA-B) and have conservation X, vary within a relatively small number of amino acids to save the activity (for example, see WO 95/13288). The mechanism by which these oligopeptides affect the activity of the immune system, unclear, in particular it is unclear how they interact with subtherapeutic doses of cyclosporine, increasing the life of allogeneic grafts.

It is also reported (Manolios, NOVEL PEPTID, patent application PCT filed on the basis of Australian applications PN 0589 and PN 0590 16 January 1995) on the effect on mediated T-cell inflammation provided by oligosacharide formula A-B-C-D-E, in which a is absent or represents 1 or 2 hydrophobic residue; b is a positively charged amino acid; C is a peptide consisting of 3 to 5 hydrophobic amino acids; D is a positively charged amino acid, and E is absent or represents up to 8 hydrophobic amino acids. Peptides that were synthesized, are Gly-Leu-Arg-Ile-Leu-Leu-Leu-Lys-Val; Met-Gly-Leu-Arg-Ile-Leu-Leu-Leu; Leu-Gly-Ile-Leu-Leu-Leu-Gly-Val; Leu-Asp-Ile-Leu-Leu-Leu-Gly-Val; Leu-Arg-Ile-Leu-Leu-Leu-Ile-Leu-Val and Leu-Arg-Leu-Leu-Leu-Lys-Val. These sequences predicted by the sequence of the transmembrane sequence of the TCR-

RELEVANT TO THIS PROPOSAL LITERATURE Buelow et al., Transplantation 59:649-654 (1995) and the references cited in this publication. Manolios et al., Nature Medicine 3:84-88 (1997) describes obtained on the basis of logic polypeptides that modulate the activity of T-cells. Publication WO 95/13288, Clayberger et al., describing peptides capable of modulating the activity of T-cells. References that describe how to design compounds with the aid of a computer using relationship patterns with activity, including Grassy et al., J. of Molecular Graphics 13:356-367 (1995); Haiech et al., J. of Molecular Graphics 13: 46-48 (1995); Yasri et al., Protein Engineering 11:959-976 (1996) and Ashton et al., Drug Discovery Today 1:71-78 (1996).

The INVENTION Offers cytomegalia peptides that are able (1) to modulate the activity of various cells of the immune system, in particular limfozitah cells, more specifically CTLs, (2) to inhibit the synthesis of inflammatory cytokines by cells capable of producing cytokines such as so effective for the treatment of conditions associated with adverse inflammatory reactions, (3) modulate the activity of hem-comprising enzymes and/or (4) to delay the onset of insulin-dependent diabetes mellitus (IDDM) in the host susceptible to IDDM by iprimary these compounds are oligopeptides, including the sequence-x-X-X-x-X-X-J-Tyr, which is a basic amino acid, J represents Gly, or an aliphatic hydrophobic amino acid containing from 5 to 6 carbon atoms, and X represents any amino acid, other than polar aliphatic amino acid, in which at least three X represent the same aliphatic non-polar amino acid, its dimer and D-stereoisomers, and this amino acid sequence can be a part of the ring. These peptides are used to suppress the activation of lymphocytes of the immune system, in particular cytotoxic lymphocytes, either separately, or in conjunction with other immunosuppressants, in particular, to increase the longevity of the graft. The described peptides are also used to suppress the synthesis of inflammatory cytokines (for example,-interferon, IL-1, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, MIPland so on), thus being useful for suppression of inflammatory reactions associated with various diseases, such as rheumatoid arthritis, septic shock, Crohn's disease, colitis, allergic reactions, autoim the Aza, synthase, nitric oxide and others , and delay the onset of IDDM patients at risk for IDDM, both in vitro and in vivo. The introduction of peptides can be performed ex vivo to be organ transplantation or in vivo, by any convenient means, including direct application or injection of peptide or nucleic acid that encodes the desired peptide in a quantity sufficient to substantially suppress lymphocyte activation, suppression of the synthesis of inflammatory cytokines and associated inflammation, inhibit the activity based on heme enzyme activity, which was previously associated with inflammatory reactions and/or to delay the onset of IDDM.

A BRIEF description of the DRAWINGS Fig. 1 depicts the conformational spatial clusters of peptide bcl-nL. Presents conformation obtained from cluster analysis of trajectories of peptide bcl-nL.

Fig. 2 depicts the projection of the trajectories of peptide in the master plan reference trajectory peptide D2.

DESCRIPTION of SPECIFIC embodiments of the INVENTION provides methods and compositions for modulating the activity of immune cells, particularly T - and b-cells and mononuclear phagocytes, more specifically the activity of the cells is th cytokines (cytokines), consequently finds application in therapeutic treatment of diseases associated with harmful inflammatory reactions, to inhibit the activity of various hem-comprising enzymes and/or to delay the onset of autoimmune diseases such as IDDM. Peptides with specific action as peptides, cytomegalia cells CTLs and NK obtained in accordance with the computer program, as described in the application corresponding to the application literature. Following the procedure described in the above publication Grassy et al., identified parameters based on known oligopeptides, for which you previously installed the ability to suppress the activity of T-cells. Cm. for example, Buelow et al., above. Conformational space required for the immunosuppressive activity was calculated as described in the above publication Yasri et al.

Using these parameters, showed that compounds with known inhibitory activity against T-cells, are found within these parameters, and it became possible to develop and test a number of new peptide compounds. New peptide compounds were with activity equal to the superior performance of izvestnyakovye), in particular, amino acids 75 to 84 and variations of this sequence, which replaced not more than two amino acids, these amino acids do not include R and Y, since the present invention does not cover such known compounds (see , for example, WO 95/13288 and the above-cited publication Buelow et al). Also known sequence based on the transmembrane region of TCR-man, consisting of this sequence and sequences that have no more than 2 mutations in this sequence. These sequences include 2 basic amino acids, and these 2 basic amino acids separated from each other by four aliphatic hydrophobic amino acids, although the application States that may be present from 3 to 5 hydrophobic amino acids. Under the mutation is meant any substitution of one amino acid by another amino acid or insertion, or deletion, each of which is counted as one mutation.

Preferably, in short sequences described here are novel peptides had two or three basic amino acids, separated from each other hydrophobic amino acids in the amount of from three to four, in particular three of hydrophobic amino the I amino acid represented an aromatic amino acid, in particular tyrosine. Especially preferably, at least one terminal amino acids cor-oligopeptides represented the amino end of oligopeptides, which may be in the form of Monomeric or oligomeric compounds.

Developed new selected peptide compounds comprising the sequence B-X-X-X-B-X-X-X-J-Tyr, which is a basic amino acid, namely Lys or AGD, in particular AGD, at least in one position, preferably in both positions, J represents Gly, or an aliphatic hydrophobic acid containing from 5 to 6 carbon atoms, in particular Gly or, and X is any amino acid other than aliphatic charged amino acid, preferably any amino acid other than polar amino acid, in which at least three X represent the same aliphatic non-polar amino acid, preferably at least four, and X represent the same aliphatic non-polar amino acids and more preferably at least all but one X represent the same aliphatic non-polar amino acid, oligomers, in particular its dimer and D-stereoisomers, and aminatta can often be increased but not more than (amount) at about 100, usually not more than about 30, more usually not more than about 20 amino acids, often no more than 9 amino acids, and amino acids have less than 25%, more usually less than 20% of the polar amino acids, more specifically less than 20% amino acids, which is charged amino acids. In addition, the terminal amino group or the carboxyl group of oligopeptides may be modified by alkylation or acylation with the formation of esters, amides or substituted amino groups, and alkyl or acyl group may have from about 1 to 30, usually from 1 to 24, or preferably from 1 to 3, or from 8 to 24, in particular 12 to 18 carbon atoms.

It also includes oligomers, including dimers of oligopeptides, which can be types "from head to head, tail to tail or tail to head", and there is no more than six repetitions of the peptide. In addition, 1 or more amino acids, up to the total number of amino acids may be D-stereoisomers.

In addition, can also be used structurally limited oligopeptides, such as cyclic peptides containing from about 9 to 50, usually from 12 to 36 amino acids, and amino acids, other than okay, not represented by amino acids. Having end cysteine, you can form a disulfide bridge, closing the ring. Alternative methods of education rings can be found in publications Chen et al., Proc. Natl. Acad. Sci., USA, 89:5872-5876 (1992) and Wu et al., Protein Engineering 6:471-478 (1993).

For the purposes of the present invention are amino acids (for the most part natural amino acids or D-stereoisomers) are divided into the following categories.

1. Aliphatic (a) nonpolar aliphatic:
Gly, Ala, Val, nL, Ile, Leu,
(b) polar aliphatic:
(1) unloaded
Cys, Met, Ser, Thr, Asn, Gln,
(2) charged:
Asp, Glu, Lys, Arg,
2. Aromatic
Phe, His, Trp, Tyr
in which Pro can be included in non-polar aliphatic amino acids, but typically not included in them. nL means norleucine, in which non-polar aliphatic amino acids may be substituted by other isomers.

Preferably, at least 3 of the six amino acids, designated as X in the peptide sequence B-X-X-X-B-X-X-X-J-Tyr, was an aliphatic amino acids, containing from 5 to 6 carbon atoms, more preferably at least 4 amino acids are represented aliphatic amino acids, containing from 5 to 6 carbon atoms, more specifically the notes, in particular non-polar aliphatic amino acid or aromatic amino acids.

Cor-sequence can be extended in any direction with the help of amino acids, which in most cases are lipophilic, namely aliphatic uncharged amino acids, and aromatic amino acids. In addition, as indicated previously, one or both, and usually one end of the oligopeptides may be replaced by a lipophilic group, usually aliphatic or aranceles having from 8 to 36, typically from 8 to 24 carbon atoms and at least two heteroatoms in the aliphatic chain, and the heteroatoms are typically, oxygen, nitrogen and sulfur. The chain may be saturated or unsaturated, preferably having no more than 3 sites, usually not more than 2 sites of aliphatic unsaturation. Conveniently, you can use commercially available aliphatic fatty acids, alcohols and amines, such as lauric acid, ministerului alcohol, stearyl amine, etc., Can be conducted by reaction of lipophilic groups with the corresponding functional group of oligopeptides in accordance with conventional ways, often during synthesis on the media, depending on the site of attachment of oligopeptides to the media.

Whom have been defined above, except U, and U is an uncharged aliphatic amino acid or aromatic amino acid, particularly a non-polar aliphatic amino acid or aromatic amino acid.

The sequence of the present invention find many uses. For research purposes they can be used to analyze physiological pathways associated with activation and deactivation of TLCs. You can combine CTLs, in particular cell lines CTLs with a known peptide targets, with peptides of the present invention, in particular tagged with a radioactive label, in the presence or in the absence of antigen presenting cells, which narrowly applicable these CTLs. After lysis performed using CTLs can be separated activated cells CTLs from dormant cells CTLs using marker CD69, which is regulated in vitro activation. The separation can be performed using FACS (cell sorting device with excitation fluorescence) and anti-CD69, tagged with a fluorescent label.

Selecting the most fluorescent cells, for example, with the highest rate in 25%, then these are lysed cells and secrete proteins associated with the markers, for example, through the Ute by electrophoresis, and then use Western blotting or other techniques with labeled peptides to identify proteins that are associated with the peptides of the present invention. Instead of a radioactive label, you can use any other type of label, usually a small organic molecule substances such as Biotin, fluorescent substance, etc., If you use Biotin, after separation, you can add avidin, and avidin labeled using the label described above.

You can also compare T-cells, which were combined with antigen presenting cells in the presence and in the absence of the peptides of the present invention. In each case, it is possible to obtain a cDNA library and use representational differential analysis, a casting or the like methods to detect differences in expression between cells that were activated in the presence and in the absence of the peptides of the present invention. You can also determine whether the reaction of certain subpopulations CTLs from the reactions of the other subpopulations in the peptides of the present invention, on the basis of their expression or lack of expression of one or more proteins, in particular proteins of the membrane surface. So you can ID manually is consistent attack tissue cells CTLs.

It is reported that peptidesdomain antigen In human leukocyte (HLA-B) associated with hsc70, which, as is known, serves as a chaperone ("companion") and is associated with a number of sequences in his role as chaperone.

Depending on their intended use, in particular for the introduction of specific mammalian hosts, the peptides of the present invention can be modified within wide limits, to replace their distribution in blood flow, reduce or increase the binding to blood components, to increase the lifetime of the peptide in the bloodstream, etc., the Peptides of the present invention may be associated with these other components using linkers that are fissile or non-digestible in the physiological environment of the blood. These peptides can be attached anywhere in the peptide, which contains a functional group such as hydroxyl, tilina, carboxyl, amino group or the like. It is desirable that the binding has occurred either in the N-end or in the end.

The peptide can be attached to a wide variety of other oligopeptides or proteins for different purposes. For example, the peptides of the present invention can the tion, moreover, antibodies can be used to identify other peptides with comparable conformation. In addition, antibodies can be used to obtain antiidiotypic antibody that can compete with the peptides of the present invention for binding site-target. These antiidiotypic antibodies can then be used to identify proteins which bind peptides of the present invention.

Alternatively, the peptides of the present invention can be expressed in combination with other peptides or proteins, making this part of the circuit, either internal or N - or C-end. By providing expression of the peptides of the present invention, it is possible to achieve different postexplosion modifications. For example, through the use of appropriate coding sequences can be achieved lipidization, such as prenisolone or monitorowania. In this situation, the peptide of the present invention must be associated with the lipid group at the end that he was able to contact a lipid membrane, such as a liposome. For the introduction of medicines, you can use liposomes, in which medication can be introduced into the loom the AI CTLs. Thus, immunosuppressants can be included in the lumen, so that immunosupressant and the peptide of the present invention could act very local.

The peptides of the present invention can be PageLayout, poliatilenaksidna provides increased life expectancy in the bloodstream. The peptides of the present invention can also be combined with other proteins such as Fc or isotype IgG, which can be complementary to contact or not to contact complementary, or toxin, such as ricin, abrin, diphtheria toxin or the like, in particular with the A-chain.

You can get these songs by obtaining the gene encoding a particular peptide or protein associated with the DNA sequence that encodes a peptide of the present invention. This gene can be entered into the appropriate expression vector, many of which are commercially available, resulting gene is then expressed in an appropriate host. Cm. Sambrook et al., Molecular Biology: A Laboratory Manual, Cold Spring. Harbor Laboratories, Cold Spring Harbor, NY, 1989.

The peptides of the present invention can be obtained by chemical synthesis or by using recombinant technologies, as described above. On sale are razlichayuttkani synthesizers natural amino acids can be replaced by amino acids of artificial origin, in particular D-stereoisomers, the side chains having different lengths or functionality, and the like. With regard to recombinant methods, it is possible to obtain a sequence of nucleic acids encoding the set of peptides of the present invention in tandem, with the help of inserted amino acids or sequence that enables the splitting up of individual peptide or dimer "from head to tail. If there is no methionine, can be used inserted methionine, which allows the splitting of individual amino acids. Alternatively, you can type consensus sequences that are recognized by specific proteases to enzymatic degradation. The specific sequence and the method for the identification of existing opportunities, economic considerations, desired degree of purity, and the like.

Chemical binding can be done with different peptides or proteins, including suitable for the formation of chemical bonds, functional groups such as amino groups to form amides or substituted amines, for example, for recovery amination, tirinya group for obrazovatelnaya peptides, having at least 2, more usually 3 and not more than about 60 lysine groups, in particular polylysine having from about 4 to 20, usually from 6 to 18 lysine units, called MAP, in which the peptides of the present invention associated with the lysine amino groups, typically constituting at least about 20%, more usually at least about 50% of the available amino groups, resulting in the formation of multipeptide product. Thus, it is possible to obtain molecules having many of the peptides of the present invention, in which the orientation of the peptides of the present invention is carried out in the same direction, and the result is a linking group that provides di - or oligomerization "from tail to tail". Alternatively, you can use other natural or synthetic peptides and proteins to get the skeleton to attach to him peptides of the present invention in the end.

In most cases, used in the composition include at least 20% by weight of the desired product, more usually at least 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes - usually at least about 99.5% of the mass against Icesave protein.

If desired, the peptide can be administered in a number of groups during synthesis or during expression, which will bind with other molecules or with the surface. So, cysteine can be used to obtain thioesters, histidine for binding to complex metal ions, carboxyl groups to form amides or esters, amino - for the formation of amides, etc., alternatively you can use a large variety of labels, as described above, including ligands for binding to antibodies or natural receptors, and peptides can be attached to the carrier or, alternatively, to another molecule. As already mentioned, the peptides of the present invention can be contacted with hsc70, which allows the isolation and purification of hsc70 from other proteins in the cell.

The peptides of the present invention can be used to modulate proliferation and/or activation of cells CTL and/or NK. By connecting peptides of the present invention with lymphocytes modulate proliferation and/or activation of CTL with antigen presenting cells, usually at least about 20%, more usually at least 40% and predpochtitelney concentration IR50for lysis in General is less than about 500 μg/ml, usually less than about 200 μg/ml and more than about 0.1 μg/ml, usually more than about 1 μg/ml.

The compositions of the present invention can be used in vitro to inhibit the lysis of antigen presenting cells, T-cells are the first target second). Thus, in those studies where it is desirable to maintain the mixture of cells in which cells CTLs were activated and destroyed antigen cells, such as macrophages or b cells, or other cells that could serve as target cells, for example cell tumors, virus-infected cells or the like, the lysis can be suppressed, allowing to maintain the cell population during the experiment.

The compositions of the present invention can also be used ex vivo. In the case of organ transplantation, in particular solid organs or certain cells, xenogenic or allogenic, donor body may be immersed in a bath with the environment, including the peptides of the present invention. Not allowed to participate present in the implant CTLs in graft versus host. In addition, during the tion CTLs recipient. Typically, the concentration of peptide in the environment varies depending on the activity of the peptide of the desired level of suppression, the presence of other compounds which adversely affect the activation of CTLs, etc., usually the concentration is in the range of from about 0.1 to 100 μg/ml, more usually in the range of about 1 to 10 μg/ml Other immunosuppressants, which may also be present include cyclosporine A, FK506, antibodies for proteins of the plasma membrane associated with graft rejection, such as antibodies to CD4, CD8, CD2, LFA-1, ICAM-1, CD28, and the like. Use subtherapeutic doses, as a rule, if they are present in an amount of not less than about 5% of normal dose and not more than about 75%, usually in the range of about 10 to 60%. Other components of the environment for bath, in which are immersed the grafts usually are components commonly used in solutions for the preservation of organs, such as HBSS. The time during which the body is supported in an environment that is usually in the range from 2 to 72 hours.

The compositions of the present invention can also be applied in vivo, by injecting the compositions of the peptides of the present invention in any convenient way. The compositions of this is 14 days prior to implantation, and preferably at least one dose administered within three days of administration. The compositions of the present invention can be introduced in the period of time starting approximately 6 hours prior to implantation, and you can continue with the introduction of the time limit under the scheme is the introduction of time after implantation, usually not more than 30 days after implantation, more usually not more than 20 days after implantation. However, after implantation of the composition of peptides of the present invention can be entered as needed, depending on the reaction of the recipient for the organ or cells. In some situations, the compositions of the present invention can be led chronically, during the whole time, until the implant is present in the host organism.

As a rule, if the peptide composition is administered directly to the host, then enter the bolus with the composition of the present invention contains in the range of about from about 0.1 to 50, more usually from about 1 to 25 mg/kg, per body weight of the host. The host may be any mammal, including farm animals, Pets, laboratory animals, primates, in particular humans. This amount is usually specified in a time-dependent half-life of the peptide, and the half-life, as a rule, is Menlow. Short time-life is acceptable, because the efficiency can be achieved by using individual doses or by continuous infusion or by applying repeated doses. The dosage of the lower part of this interval, and even lower doses may be used in cases where the peptide has an increased half-life or if it is applied in the form of a depot, such as particles, including slow-release composition, is introduced into the matrix, which stores the peptide over an extended period of time, for example in collagen matrix, or by using a pump that continuously delivers the peptide in an extended period of time with substantially constant speed, and etc.

In addition to the introduction of the peptide compositions of the present invention directly into the cell culture in vitro, application to solid bodies or specific cells ex vivo, or the introduction of in vivo host, representing a mammal, they can be entered or filed molecules of nucleic acid (DNA or RNA) encoding the peptides of the present invention, which provides an effective source of peptides of the present invention for the desired application. In most cases icesto well known expressing plasmids (see Maniatis et al., above) and/or a viral vector, preferably an adenoviral or retroviral vectors (see , for example, Jacobs et al., J. Virol., 66:2086-2095 (1992), Lowenstein, Bio/Technology, 12: 1075-1079 (1994), and Berkner, Biotechniques, 6:616-624 (1988)), under the transcriptional control of regulatory sequences, the function of which is to stimulate the expression of nucleic acids in the respective environmental conditions. These are based on nucleic acid carriers can be submitted directly to the tissue graft ex vivo (e.g., viral infection of cells ex vivo for transplant) or to the desired site in vivo, for example, by injection, catheter, etc., or, in the case based on virus carriers, you can enter the system. Alternatively, you can use tissue-specific promoters, with which interest peptide is expressed only in specific tissue or cell type of choice. Methods for such based on the nucleic acids of media using recombinant technologies well known in the art, as well as the techniques based on nucleic acid carriers for the production of peptides, both in vitro and in vivo.

Transplantation can vcloud or other authority, and cells, such as-insular cells, bone marrow cells or other cells, and the body or the cells may be allogeneic or xenogeneic, in particular, when one or more antigens of class I or class II MHC (MHC, major histocompatibility complex) in the donor and recipient are different.

The peptides of the present invention, individually or in the form of conjugates, or represents a nucleic acid media encoding such peptides can be obtained in the form of dosage forms in a pharmaceutically suitable environments, such as, for example, saline, SFR (phosphate buffered saline) solution, an aqueous solution of ethanol, glucose, propylene glycol or the like, or in the form of solid dosage forms in the respective fillers, as a rule, a pharmacologically effective dose. The concentration of the peptides or their coding nucleic acid is determined empirically in accordance with conventional methods for a particular purpose. Dosage forms may include bactericidal agents, stabilizers, buffers or the like. The number entered to the owner, varies depending on what is introduced, from the purpose of introduction, such as prevention is at the introductions and the like, and this number can be determined empirically by experts in the field of technology. To increase the length of time half-life of the peptide of the present invention or the peptide conjugates of the present invention, the peptides can be enclosed in capsules, to enter into the lumen of liposomes, prepared in the form of a colloid or you can use other conventional methods, providing increased life expectancy peptides ex vivo or in vivo.

The peptides of the present invention are able to suppress the production of cells of inflammatory cytokines. Inflammatory cytokines, inhibiting peptides of the present invention, include, for example, necrosis factor tumor cells, including factor-necrosis of tumor cells (TNF-), interferons, including-interferon (INF-), interleukin IL-l, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, 1L-16, MIPl-, chemokines, hematopoietic growth factors and the like, both in vitro and in vivo. Therefore, the peptides of the present invention is applicable to both preventive and therapeutic suppression of inflammatory reactions associated with various diseases, tehicheskie reaction, atherosclerosis, infection, as well as many other situations where it is desired anti-inflammatory reaction.

The compositions of the present invention is also capable
to modulate the activity of hem-comprising enzymes both in vitro and in vivo. As shown below, the peptides of the present invention mimic porphyrinogen structure, capable of modulating the activity of hem-comprising enzymes such as heme oxygenase (TH), different isoforms of nitric oxide synthase (NOS), cyclooxygenase, guanilatziklazu and the like. Therefore, the compositions of the present invention can be used in situations where it is desirable to adjust the expression of hem-comprising enzyme, such as hemoxygenase. In this regard, it is known that heme oxygenase is involved in the pathways of metabolism other than modulation impolitically activity. Therefore, when adjusting the expression of hemoxygenase will be adversely impacted on the ways of metabolism involving hemoxygenase. See, for example, Willis et al., Nature Medicine, 2:87-89 (1996).

In addition, it is reported that hem-comprising enzymes such as heme oxygenase, are a factor of the inflammatory response and may have anti-inflammatory effect. Therefore, various physiological processes, by comparing the cellular responses in the presence and in the absence of the peptides of the present invention. The peptides of the present invention can also be used in vivo to reduce the inflammatory reaction associated with septic shock, Crohn's disease, colitis including ulcerative colitis and mucous colitis, rheumatoid arthritis, atherosclerosis, re-perfusion, infection and the like. Description of the use of the peptides of the present invention to modulate impolitically activity substantially applicable for these indications.

The peptides of the present invention can also be used to delay the onset of autoimmune disease related to the mammalian subject at risk for the development of autoimmune diseases. The peptides of the present invention is particularly applicable to delay the onset of insulin-dependent diabetes mellitus (IDDM), rheumatoid arthritis or systemic lupus erythematous in a mammal at risk of developing these diseases. Description of the use of the peptides of the present invention to modulate impolitically activity substantially applicable for these indications.

Following the EXPERIMENTAL PART
A computer program used to predict and development immunosuppressive activity of peptides and pseudopeptides, was carried out as follows.

1. METHODOLOGY
Based on the initial set of experimental data obtained for peptides exhibiting or not exhibiting immunosuppressive activity, the following was obtained.

i. A consensus sequence containing amino acids required for activity and that helps to get new library of peptides or pseudopeptides.

ii. A set of physico-chemical and topological properties associated with the activity, and converted into a set of constraints using the methods of mapping variables (Grassy et al. , J. of Molecular Graphics, 13:356-367 (1995)).

2. MAPPING VARIABLES
The method is based on physico-chemical and topological constraints obtained on the basis of the results of the initial data set.

PHYSICO-CHEMICAL CONSTRAINTS
The method requires the determination of physico-chemical constraints, defined as the ranges of properties for the specified biological activity. The calculation method used to determine the set of constraints is called the method of mapping variables and describes the lower inogo or percentage) of active and inactive molecules as a function of the values of these parameters. The superposition of all graphs (activity-property) specifies, for certain parameters, the limiting values (low and/or high), which are necessary to obtain active compounds. This graphical method identifies qualitative nonlinear dependence between the activity and molecular properties. With regard to the properties associated with the interactions of the ligand of the receptor, it was clearly established that the existence of a strict contingency indicators of adaptation to the receptor, implies the inclusion of certain structural and physico-chemical properties. This method gives a simple rule that can be used to predict the activity of unknown products. A graphical representation showing the number of cases of successful execution rules in relation to the number of cases of violation of the rules allows you to compare the distribution activity indicators for all the studied set of molecules.

3. PHYSICOCHEMICAL AND TOPOLOGICAL PARAMETERS USED TO DETERMINE THE LIMITATIONS ASSOCIATED WITH IMMUNOSUPPRESSIVE ACTIVITY OF PEPTIDES AND PSEUDOPEPTIDES.

LIPOPHILICITY
The lipophilicity of peptides were expressed as Tr (where P predstave logP can be calculated using TSAR 2.31, using increasing atomic logP values defined by Goutom and others (Ghose et al., J. Chem. Inf. Comput., 29:163 (1989). As shown by analysis of the initial data set, the lipophilicity of immunosuppressive peptide should be-6,85.

TOPOLOGICAL INDICES
The BALABAN INDEX (Balaban, Chem. Phys., 89:399 (1982))
The Balaban index, calculated for a connected molecular graph (N depressed), calculated as follows:

where M is the number of edges in the graph,represents the cyclomatic number of the graph, i.e., the minimum number of edges that must be removed before G will be acyclic, and Di=Dii(j=1) represents the distance matrix of the shortest path between two vertices of the graph.

The MOLECULAR VOLUME
The molecular volume calculated by taking the standard value of the radius van der Waals forces for each element. This calculation is performed on the extended conformation of the peptide.

ELLIPSOIDAL VOLUME
This amount is calculated after determining the three components of the moment of inertia of the molecule, taking average values of the atomic mass for sostavlyaya ABILITY
Molar refraction ability are estimated using values of atomic molar refractive ability, designed Ghose et al. (cited above).

DIPOLE MOMENT
This parameter is calculated from the extended conformation of the peptides. The total dipole moment for the molecule is expressed in units of Debye.

= eriqi,
where riis the distance from atom i to the origin, and qiis the charge of atom i. The charges of the atoms are estimated using the method of the Charge-2 (Abraham and Smith, J. Comput. Aided Mol. Design, 3:175-187 (1989)).

INDEX KIRA Chi V 4
This index represents one of the indices of connectivity, designed by Cyrus L. B. Kier). Index Kira Chi V 4 shall be calculated in several stages (N included).

A. Definition and numbering of all paths of length 4 on the molecular graph of the peptide.

B. Calculation of each path of length 4 of the following values:
Cvs= P[(vj)]-0,5,
for j=1,4, wherei=Zi-hiis determined for the atom as the difference between the total number of valence electrons Ziand the number of hihydrogen atoms associated with atom i.

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INDEX KIRA KAPPA ALPHA
Index Kira Kappa alpha 1 (K1)
If a represents the total number of atoms of the molecule (N included), then K1equal to the following:

with:

riis a covalent radius of atom i, and rCsp3covalent radius of carbon sp3P1represents the total number of paths of length = 1 along the molecular graph of the studied peptide.

Index Kira Kappa alpha 2 (K2)
If a represents the total number of atoms of the molecule (N included), then K2equal to the following:

with

riis a covalent radius of atom i, and rCsp3covalent radius of carbon sp3, R2represents the total number of paths of length = 2 along the molecular graph of the studied peptide.

FLEXIBILITY Phi
Based on the above formulas, the flexibility of the molecule can be determined as follows:
Phi = (K

The NUMBER of ATOMS AND GROUPS
As restrictions used the number of the following types of atoms:
- the total number of oxygen atoms in the peptide,
- the total number of nitrogen atoms in the peptide.

As restrictions used the number of the following groups:
- the total number of ethyl groups,
- the total number of hydroxyl groups.

4. The limit VALUES
CREATING LIBRARIES of PEPTIDES OR PSEUDOPEPTIDES
Since the consensus sequence AGD-x-X-X-AGD-x-X-x-X-Tight, in which X represents an amino acid, which is defined in the previously shown a similar formula was calculated above physicochemical and topological parameters and determined whether these parameters are within the limits defined by the original data set. For example, starting with X= Leu, nLeu, Trp, Tight, Gly and Val, have created a library consisting of 279936 molecules, and only 26 of them met the conditions required restrictions.

The ranges of the properties required for obtaining biological activity, are summarized in table 1.

8. The characterization of the CONFORMATIONAL SPACE INVOLVED IN the IMMUNOSUPPRESSIVE ACTIVITY of PEPTIDES AND PSEUDOPEPTIDES
In the structure was first introduced Broto and other (Broto et al. , Eur. J. Med. Chem., 19:66-70 (1984)). This vector is essentially discretized distribution of distances obtained from the matrix of interatomic distances of the molecule. The first component of this vector (A ) is equal to the number of atoms of structures, and other components, And1. . .Andnare determined by the number of pairs of atoms that are separated by a distance within the interval defined by a lower bound (n-1)Diwhere n represents the order of an element discretization of the vector, and Di- the distance increment. Similarly, we can calculate the distribution of the atomic characteristics of R. In this case, the weighted component autocorrelation ARnis obtained by summing the products of the values of the characteristics of the P atoms i, j, having a distance from each other, belong to the interval of distances [(n-1)DinDi]. Then determine the number of components of the vector from the equation nmax=(Dmax/Di)+1, where Dmaxrepresents the largest interatomic distance in the structure.

Vector autocorrelation shows several useful properties.

Using this vector can significantly reduce the amount of data conformation. General conformation describes how restricted is about to compute and store this vector during the simulation of molecular dynamics, and in this process there is a reduction of the storage size in comparison with the classical conservation of the full set of matrices of distances that allows to simulate much longer than usual.

- Vector autocorrelation conformation is transitional and rotational invariant, and independent of the number of atoms in the molecule.

- This vector are sensitive both to small and to large changes in conformation: the more changes the conformation, the more modified components of the vector. The sensitivity depends on the increment of the distance selected for the calculations, but the increment from(small molecules) to(macromolecules) is a good choice for normal simulations (Yasri et al., Protein Engineering, 11:959-976 (1996)).

It is possible to analyze only part of the structure or only a specific subnavbar atoms of this structure, for example, With2in proteins, the N atoms, heavy atoms, etc., the Vector is completely determined by the knowledge of the structure, so you can make the comparison of different structures using this vector without any reference.

The ANALYSIS of MOLECULAR DYNAMICS USING VECTOR AUTOCORRELATION 3-D
In relation to the peptide HLA-B2702. the denim peptides have performed molecular dynamics simulation using the AMBER 4.1. The simulation of one nanosecond dynamics creates a set of 103conformations (one conformation in the PS). For each conformation was calculated vector autocorrelation 3D using TSAR, with the distance increment inand the entire set of conformations retained in the form of vectors of the 3D autocorrelation versus time matrix (103xn).

The aim of this work was to determine the conformational space, responsible for the immunosuppressive activity by comparing the conformational spaces of active and inactive peptides, using the techniques described in the references listed in the section Relevant to this proposal literature".

STATISTICAL ANALYSIS
CLUSTER ANALYSIS
In order to compare different conformations, determined the distance matrix between all of these conformations in the hyperspace defined by components of their unweighted vector autocorrelation 3D. The more similar the structure of the two compounds, the shorter the distance between them. This method provides the possibility to quantify the rigid molecular compliance. When using the start information as a reference numerical value of this distance is made by a multivariate statistical method of data analysis suitable for the representation of molecules in the hyperspace of their properties (molecular descriptors). PCA can be used to reduce the large number of descriptors to a smaller number of synthetic orthogonal variables, obtained from a linear combination of the original descriptors. This method preserves most of the original information. The original variables were normalized and was calculated diagonalizable covariant matrix using the classical transformational programs Jacobi (Jacobi). Components autocorrelogram vector 3K provide a good description of the structure of 3K different conformations, but it is inconvenient to work because they contain too much data, which does not allow for easy visualization. PCA can reduce the dimensionality of the data to view 2D or 3D, which has the maximum number of source information. When using PCA immunosuppressive peptides exhibit a well-defined total conformational space. All peptides are able to achieve these conformational indicators, can show immunosuppressive activity.

The coordinates of the CONFORMATIONAL SPACE of BIOACTIVE Conformality bcl-nL, in which the peptide bcl-nL has the amino acid sequence Arg-nL-nL-nL-Arg-nL-nL-nL-Gly-Tyr and where nL represents norleucine (see below). Shows patterns were obtained by applying cluster analysis on the whole trajectory of the peptide bcl-nL.

The MAIN CONFORMATION of the PEPTIDE bcl-nL
Structural properties investigated the main conformations of the peptide bcl-nL (Fig. 1, (1), (2), (3), (4) and (5)) and its conformational space are summarized in table 2. These properties relate to the coordinates in three-dimensional space defined by the first three principal components (PCA coordinates), and the radius of the circular motion (Rg).

The CONFORMATIONAL SPACE of the ACTIVE PEPTIDES
The trajectory of the D2 peptide (amino acid sequence Arg-Val-Asn-Leu-Arg-Ile-Ala-Leu-Arg-Tyr) was described using the autocorrelation method and 3D data were analyzed using principal components analysis. This gave the opportunity to obtain the master plan defined by the first 2 principal components, which contains all conformations examined the trajectory. The trajectory of the D2 peptide used as a reference trajectory and all calculated trajectories projected in the master plan (Fig. 2).

Immunosuppressive peptides show Ho the PCA:
PC1: minimum = -2,0; max = 2,0
RS: minimum = -2,0; max = 1,0
RS: minimum = -1,0; max = 1,0
The peptides obtained in the form of compositions are given in table 3.

EXAMPLE 1. ANTIPROLIFERATIVE ACTIVITY b-PEPTIDES
Adults 6-8-week-old male mice of lines C57BL6/J (B6, H-2d), 1b/s(N-2d) and CBA/JH-2k) were purchased from Jackson Laboratory, Bar Harbor, ME. They were kept and maintained in the laboratory for the animals at SangStat Medical Corporation according to the guidelines of the National Institute of health (USA) and in accordance with the rules of the Department of health.

The peptides synthesized in synt:em (Nimes, France) using a peptide synthesizer and methods Fmoc-chemistry. All peptides were synthesized in the form of amides, and then was converted into the acetate salt. Peptides were purified using preparative HPLC with a reversed phase analytical HPLC with reversed phase showed that they are more than 95% homogeneous. Amino acid content was confirmed by analysis of amino acids. Before using the peptides were first dissolved in 1 volume of DMSO (Sigma), and then added to 99 volumes of culture medium. The final concentration of DMSO in culture was not greater than 0.25%.

Suspensions of spleen cells were obtained by the de and, finally, resuspendable in medium RPMI-1640 with 10% FBS (serum fetal cow)(R-10) or in medium without serum AIM-V (Gibco, Grand Island, NY).

The spleen cells isolated from mice of CBA, then subjected to stimulation (2105per well) monoclonal antibody anti-D3 (Pharmagen, San Diego), at final concentrations from 0.1 to 1 µg/ml in 96-well round-bottom microcultural plates (Nunc, Denmark). At the beginning of the cultivation was added b-peptides at various concentrations. Cells were incubated for 3 days at 37oC and 5% CO2. 24 hours before collection grown in cell culture in each well was added 1 MK Ci[3H]-TdR (Amersham, Arlington Heights, IL). Then the cells were collected using a harvester (provisions for collecting cells) brand Filtermate 196 Harvester (Packard, Downers Grove, IL) and measured the degree of incorporation of thymidine using a scintillation counter for microplate brand TopCount (Packard).

The results obtained in this research showed that the solution SFR/DMSO without peptide and control peptide 2705 (amino acid sequence Arg-Glu-Asp-Leu-Arg-Thr-Leu-Leu-Arg-Tyr) had no effect on the proliferation of T-cells, and b-peptides inhibited the proliferation of T-cells, while the value of the ability to inhibit the proliferation of T cells.

EXAMPLE 2. INFLUENCE b-PEPTIDES TO CYTOTOXIC ACTIVITY of T CELLS
To analyze the effect of peptides on the cytotoxic activity of T cells, received effector cells CBA to B6, after 6 days of cultivation 4106the spleen cells with IAS 5106treated with mitomycin of B6 spleen cells in the wells of a 24-hole tablet (Nuncion Delta, Nunc, Denmark) in RPMI-1640 with 10% FBS. Then the effector cells were collected and washed. As target cells used EL4(H-2b), cell lymphomas of mice induced in C57BL/6N. Cultured cells EL4 supported and passively every three days (51SG) in 20 μl for 1 hour at 37oC. Then the effector cells (E) and target cells (T) were placed in a V-shaped culture Cup (Nunc, Denmark) at the ratios of E:T, components 3:1, 10:1, 30:1 and 100:1 respectively. Peptides were diluted environment R-10 to working concentrations and added to the beginning of the 4-hour incubation period. To determine the maximum output in individual wells was added 1% Triton X-100. Then the Cup was centrifuged for 2 minutes in order to increase cell contact before a 4-hour incubation period. After incubation was taken in 75 μl of adosados is Kaplanov brand TopCount. The degree of cell lysis was calculated using the formula below:

where CPM is the number of counts per minute.

The results of these analyses showed that the control peptide 2705 at concentrations up to 100 µg/ml had no effect on mediated T-cell lysis of target cells, while b-peptides inhibited mediated cell lysis, and the degree of inhibition was dose dependent. Premaxillae inhibition of cell activity of CTLs induced b-petidomo, was observed at a concentration of about 0.5 μg/ml.

EXAMPLE 3. INFLUENCE b-PEPTIDES ON ALLOGENEIC GRAFTS in vivo
Immunosuppressive activity b-peptides was evaluated on the model vascularizing completely incompatible allograft heart of a mouse. Specifically, abdominal heterotopic heart transplantation was performed as previously described in the work of Ono and Lindsey, J. Thoracic Cardiovasc. Surg., 7:225 (1969). The mice CBA, were recipients of hearts C57B1/6, were daily injected with varying doses of the peptide, after an organ transplant. The peptides were dissolved in DMSO and diluted in SFR (final concentration of DMSO was 10%) before intraperitoneal injection. Animals Wali daily by direct palpation and rejection was defined by the cessation of palpable contractions of the heart. The statistical significance of the duration of survival of cardiac allograft was calculated using test Mann-Whitney.

The results of these tests showed that the control peptide 2702.75-84 (amino acid sequence AGD-Glu-Asn-Leu-Arg-Lle-Ala-Leu-Arg-Tyr), introduced at a dose of 80 mg/kgday (day 0-9), contributed to the prolongation of survival of cardiac allograft to 10.72.6 days compared to 81.4 days in control animals that received SFR/DMSO (p<0,01). Introduction the control peptide 2702.75-84 at a dose of 40 mg/kgthe day had no significant effect on graft survival compared to the control variant. In contrast, however, the introduction b-peptides in such a low dose of 1 mg/kgthe day resulted in a significant increase in the duration of survival of cardiac allograft, and 50% of the grafts remained alive for more than 28 days. Thus, these results show that b-peptides have immunosuppressive activity, sufficient to increase the duration of survival of grafts in a mammal.

EXAMPLE 4. Sposobnosti analyses on the protein binding. Specifically, the synthesized peptide 2702.75-84 in biotinylating form attached to the N-end Biotin, through six of the carbon atoms of the spacer. On the surface of the tablets for ELISA (enzyme-linked immunosorbent assay) (Nunc Maxisorb, Nunc, USA) was applied 100 ng/ml recombinant hsc70 (Stressgen, Victoria, Canada) in 100 mm Na-citrate buffer with pH 4.0 and left until the next day at a temperature of 4oC. After that, the remaining binding sites were blocked by incubation cups SFR/0.1% tween-20 (PBS/Tween, Sigma) for 2 hours at room temperature. Unbound material was removed by three times washing of cups mixture SFR/twin room. After the addition of the biotinylated 2702.75-84 in the mixture SFR/twin/1%DMSO cups were incubated for 2 hours at room temperature, washed three times, then incubated with 0.1 μg/ml streptavidin conjugated to horseradish peroxidase (streptavidin-HRP) (Jackson ImmunoResearch Laboratories, West Grove, PA) and again washed. Linked streptavidin-HRP was detected using 3 mg/ml o-phenylenediamine (OPD, Sigma) in substrate buffer (SangStat, Menlo Park, CA). The reaction was stopped by adding 1M Hcl and measured the absorbance (optical density OP-OP), using the device for reading of IFA tablets. The binding of unlabeled peptide CSOs b-peptide and then incubated in the tablets coated with hsc70 for 3 hours at room temperature. Then determined associated biotinylated 2702.75-84, as described above.

The results of these experiments showed that affinity chromatography using the peptide 2702.84-75-75-84 (inverted dimer) led to the isolation of hsc70 and hsp70 (jitsukawa protein). After incubation of the plates to the IFA on the surface of which was caused hsc70 with biotinylated peptide 2702.75-84, the authors present invention was observed in a dose-dependent binding of this peptide with hsc70. Binding of the biotinylated peptide 2702.75-84 with hsc70 may also be inhibited by addition of increasing concentrations of peptide 2702.75-84. Premaxillae inhibition (IR50) was observed at 7,03.0 mm, such IR50was observed for b-peptides (IR50=2.5 to 10 μm), suggesting that b-peptides effectively communicate with hsc70.

EXAMPLE 5. INFLUENCE b-PEPTIDES ON HEMOXYGENASE AND OTHER hem-comprising ENZYMES
Influence b-peptides on hsc32 (hemoxygenase) was assessed by measuring the activity of hemoxygenase (TH) in the presence and in the absence of b-peptides. Specifically, samples of mouse spleen homogenized on ice to lyse buffer Tris-C1 (pH 7.4) containing 0.5% Triton X-100 and protease inhibitors. Samples Zam is the source for all activity measurements. Obtained from the liver of rats biliverdine was purified by the method described in the publication Kutty and Maines, J. Biol. Chem., 256:3956 (1981). The activity of CS was measured by mixing 100 μl of the homogenate spleen 0.8 μm NADPH, 0.8 mm glucose-6-phosphate, 1.0 unit G-6-P-degidro-genaz, 1 mm MDS2and 10 ál biliverdine at 4oC. the Reaction was initiated by addition of hemin (20 μl of a 2.5 mm solution). The reaction mixture was incubated at 37oC in the dark for 30 minutes. At the end of the incubation period, all insoluble materials were subjected to centrifugation and analyzed the supernatant concentration of bilirubin using a modified method of Gilman and Beyer (Hillman and Beyer, Z. Klin. Chem., 5:92 (1981) (using a diagnostic kit 552 Sigma). Control options include samples of the spleen in the absence of an NADPH-generating system and all components of the reaction mixture in the absence of spleen homogenates.

The results of these experiments showed that, compared with inactive control peptide 2705.75-84, bc-peptides (100 μg/ml) inhibited the activity of TH more than 50%. Thus it was found out that, apparently, b-peptides are able to inhibit the activity of hemoxygenase.

After ustanavlivaetsya enzymes, such as synthase nitric oxide (NOS). Specifically, b-peptide and control peptide 2702 explored in the analysis of the activity of the enzymes for their ability to inhibit three different isoforms of NOS (neuronal NOS, endothelial NOS, and inducible cytokine NOS) in vitro. The results of these experiments showed that b-peptides are able to inhibit NOS with a significantly lower rate IR50than the rate of control peptide 2702. Thus it was found out that, apparently, b-peptides mimic porphyrinogen patterns that particularly affect the activity hem-comprising enzymes, and this was confirmed by computer simulation.

EXAMPLE 6. INDIRECT b-PEPTIDES inhibition of the SYNTHESIS of INFLAMMATORY CYTOKINES
In order to determine the influence of b-peptides in the synthesis of inflammatory cytokines by cells of the macrophage RAW264.7 in culture with 10 μg/ml of bacterial lipopolysaccharide (LPS) was affected in such a way as to stimulate the production of their inflammatory cytokine factor-necrosis of tumor cells (TNF-(see Alleva et al., J. Immunol., 153:1674 (1994) and Tonetti et al., Biochem. Biophys. Res. Comm., 230:636-640 (1997)), either in the absence or in the presence of from 1 to 100 μm)lnyh supernatant fluids using ELISA. Without addition of LPS RAW264 cells.7 was produced detectable amounts of TNF-.
The results obtained in these experiments showed that while the control peptide D2RP (amino acid sequence Arg-Val-Asn-Leu-Pro-Lle-Ala-Leu-Arg-Tyr) has not shown the ability to inhibit the production of TNF-cells of the macrophage RAW264.7, b-peptides inhibited the production of TNF-and the degree of inhibition was dose dependent. Thus, b-peptides will be used in the suppression of the synthesis of inflammatory cytokines, thus being a valuable tool for the treatment of inflammatory and associated with inflammatory diseases.

EXAMPLE 7. ACTION b-PEPTIDES WHEN USING THEM ON the model of SEPTIC SHOCK IN ANIMALS
The introduction of LPS to mice is a good model of septic shock in animals (see Otterbein et al., Amer. J. Physiol., 272 (2):1 (1997), Albrecht et al., Hepatology 26:1553-1559 (1997), Haziot et al., J. Immunol. 154: 6529-6532 (1995) and Otterbein et al., Amer. J. Respir. Cell Mol. Biol., 13: 595-601 (1995)). In order to obtain more evidence of the usefulness b-peptides for the treatment of inflammatory reactions and inflammatory conditions (such as septic shock), the authors were influenced by b-peptides on viziunile or mannitol. After 16 hours after administration of the peptide in the mannitol or mannitol mice were injected with an injection of 100 mg/kg LPS and twice a day tracked the survival of mice.

The results of these experiments showed that while all mice that were injected control peptide 2705 in mannitol or mannitol, died the next day after administration of LPS, more than 50% of mice treated with b-peptide, were living on the second day after administration of LPS, and more than 25% of mice treated with b-peptide, were living on the third day after administration of LPS. Thus, these data show that b-Petey effective for the treatment of inflammatory conditions such as septic shock.

EXAMPLE 8. INTRODUCTION b-PEPTIDES BY means of GENE TRANSFER in vivo INCREASES the SURVIVAL of MURINE HETEROTOPIC CARDIAC TRANSPLANTS
Next, the authors set out to determine whether local delivery b-peptides mediated by plasmid transfer genes to improve the survival of grafts in vivo in the mouse model of heterotopic heart transplant. Specifically, neonatal heart donor C57BL/6 subcutaneously transplanted into the outer ear of mice recipient CBA/J. At the time of transplantation directly into the allograft of spianato was determined by monitoring of the electrocardiogram and the rejection was determined on the basis of cessation of electrical activity of the heart. Survival of grafts expressed in days (averagethe standard error). Statistical significance was determined using unpaired t-test t-test.

Direct injection of 1 μg of control peptide 2702 in the allograft is not extended term survival (13,30,75, against 13.90.9 in the control without injection of the peptide), and injection of 400 μg of peptide 2702 extended term survival (22,00,58). Injection of 20 μg of plasmid DNA, the coding control peptide 2702, a more extended term survival of the graft - to 30.3of 1.03. Similar results were obtained for another plasmid that encodes a peptide bcl (29,04,08), whereas it was not observed significant increase in survival time when using a plasmid that encodes a control peptide 2705, not having immunomodulatory activity in vitro or in vivo (16,50,96).

EXAMPLE 9. INFLUENCE b-PEPTIDES TO DELAY the onset of INSULIN-dependent DIABETES MELLITUS (IDDM)
Since in the present description it is shown that the peptides of the present invention are immunomodulatory, the inventors next goal was opredelili autoimmune diseases in General, the authors determined the ability b-peptides to delay or inhibit the onset of IDDM in vivo. Specifically, 20 mg/kg b-peptide was administered intraperitoneally to 6-week old female mice without diabetes (LMI), while control mice not injected or injected inactive peptide compound. The above options were repeated every week. In experimental animals once a week was determined by the glucose in the blood. The onset of IDDM in experimental animals was determined by the level of blood glucose greater than 200 mg/DL.

The results of the above experiments showed that 70 - 80% LMI control mice not treated with the peptide, IDDM developed by the age of 22 weeks. No difference was observed between the control groups of animals which either received no peptide, or has been inactive control peptide. However, for the animals, which were injected b-peptides, only one animal developed IDDM by the age of 16 weeks, and all the rest of the experimental animals did not develop IDDM by the age of 24 weeks. Thus, these results show that the introduction of b-peptides effective to delay the onset of IDDM in vivo.

From the above results it is evident that the compositions and methods of the present invention provide significant inhibition of cytotoxicity of CTLs. You the of lysis compared with a previously used oligopeptides. The use of the compositions of the present invention provides a significant advantage in that it requires a smaller number of oligopeptides to achieve therapeutic levels, as well as in suppressing hemoxygenase activity.

All publications and patent applications mentioned in the present description, are included as references to the same extent as if each individual publication or patent application would be specifically and individually indicated that they are included as a reference.

Now, when the invention is fully described, the specialists of ordinary skill in the art it is obvious that you can make many changes and modifications of the present invention, which do not go beyond being or scope of the attached claims.


Claims

1. Oligopeptide defined by the parameters given in table 1, comprising the amino acid sequence of
B-X-X-X-B-X-X-X-J-Tyr,
which is Lys or Arg;
X represents any amino acid, other than charged aliphatic amino acid or its D-isomer;
J is Gly, Lys or Arg, where these amino acids predstavljaet (a) a naturally occurring sequence1domain antigen In human leukocyte (HLA-B) 75-84, (b) a naturally occurring sequence of the transmembrane sequence- chain T-cell receptor of human rights and (in) consistency, which is the mutant with respect to either (a) or (b) having no more than two mutations as an active part of the above oligopeptides.

2. Oligopeptide under item 1, with the specified Oligopeptide contains not more than 20 amino acids and is a monomer, dimer or includes part oligopeptides rings.

3. Oligopeptide under item 1, in which In - Arg; X represents an aliphatic non-polar amino acid containing from 5 to 6 carbon atoms, including norleucine, an aromatic amino acid or its D-isomer, and in the specified Oligopeptide contains at least 3 identical nonpolar aliphatic acid; and J is Gly or Arg.

4. Oligopeptide under item 3, in which at least 5 amino acids, defined as X, are valine, leucine or norleucine, and any of the other acids, defined as X, is Thr or Tight.

5. Oligopeptide under item 4, in which at least 5 amino acids, defined as X, are the same.

6. Oligopeptide under item 1, including the(b) Arg-Val-Leu-Leu-Arg-Leu-Leu-Leu-Gly-Tyr;
(c) Arg-Ile-Leu-Leu-Arg-Leu-Leu-Leu-Gly-Tyr;
(d) Arg-Leu-Val-Leu-Arg-Leu-Leu-Leu-Gly-Tyr;
(e) Arg-Leu-Ile-Leu-Arg-Leu-Leu-Leu-Gly-Tyr;
(f) Arg-Leu-Leu-Val-Arg-Leu-Leu-Leu-Gly-Tyr;
(g) Arg-Leu-Leu-Ile-Arg-Leu-Leu-Leu-Gly-Tyr;
(h) Arg-Leu-Leu-Leu-Arg-Val-Leu-Leu-Gly-Tyr;
(i) Arg-Leu-Leu-Leu-Arg-Ile-Leu-Leu-Gly-Tyr;
(j) Arg-Leu-Leu-Leu-Arg-Leu-Val-Leu-Gly-Tyr;
(k) Arg-Leu-Leu-Leu-Arg-Leu-Ile-Leu-Gly-Tyr;
(l) Arg-Leu-Leu-Leu-Arg-Leu-Leu-Val-GIy-Tyr;
(m) Arg-Leu-Leu-Leu-Arg-Leu-Leu-Ile-Gly-Tyr;
(n) Arg-Trp-Leu-Leu-Arg-Leu-Leu-Leu-Gly-Tyr;
(a) Arg-Leu-Trp-Leu-Arg-Leu-Leu-Leu-GIy-Tyr;
(p) Arg-Leu-Leu-Trp-Arg-Leu-Leu-Leu-Gly-Tyr;
(q) Arg-Leu-Leu-Leu-Arg-Trp-Leu-Leu-Gly-Tyr;
(r) Arg-Leu-Leu-Leu-Arg-Leu-Trp-Leu-Gly-Tyr;
(s) Arg-Leu-Leu-Leu-Arg-Leu-Leu-Trp-Gly-Tyr;
(t) Arg-Tyr-Leu-Leu-Arg-Leu-Leu-Leu-Gly-Tyr;
(u) Arg-Leu-Tyr-Leu-Arg-Leu-Leu-Leu-Gly-Tyr;
(v) Arg-Leu-Leu-Tyr-Arg-Leu-Leu-Leu-Gly-Tyr;
(w) Arg-Leu-Leu-Leu-Arg-Tyr-Leu-Leu-Gly-Tyr;
(x) Arg-Leu-Leu-Leu-Arg-Leu-Tyr-Leu-Gly-Tyr;
(y) Arg-Leu-Leu-Leu-Arg-Leu-Leu-Tyr-Gly-Tyr;
(z)-Arg-Nle-Nle-Nle-Arg-Nle-Nle-Nle-Gly-Tyr.

7. Oligopeptide under item 1, comprising the Oligopeptide under item 6.

8. Oligopeptide under item 7, which has no more than 100 amino acids.

9. Oligopeptide under item 8, which is not more than 30 amino acids.

10. Oligopeptide under item 9, which has no more than 20 amino acids.

11. The method of suppressing the activation limfozitah cells, and the method includes connecting these cells with the overwhelming activation of a number of compounds, including Oligopeptide according to any one of paragraphs.1-6, and activation of these limfozitah cells is suppressed.

12 authority or viable cells, other than limfocitna cells.

13. The method according to p. 11, in which the specified stage connection includes providing contact these limfozitah cells with a nucleic acid molecule that encodes a specified Oligopeptide, and specified Oligopeptide is expressed by the specified nucleic acid molecule.

14. The method according to p. 13, where this nucleic acid molecule is contained in the virus.

15. The method according to p. 11, in which these limfocitna cells are cytotoxic T-lymphocytes.

16. The method of transplantation of donor organs or cells of mammals, other than part of a viable organ recipient, representing a mammal, which includes the capture of these donor organ or cells from the donor and the implantation of the specified recipient, and the method includes at least the following stages: (a) the conjunction of the specified organ or cells prior to implantation in the specified related to the recipient mammal with an overwhelming activation of a number of compounds, including Oligopeptide according to any one of paragraphs. 1-6 or (b) the introduction of specified related to the mammalian patient within a time period lasting from the moment before implantation and before the PTO what about the Oligopeptide according to any one of paragraphs.1-6.

17. The method according to p. 16, in which the specified stage connection includes providing contact specified organ or cells with a nucleic acid molecule that encodes a specified Oligopeptide, and specified Oligopeptide is expressed by the specified nucleic acid molecule.

18. The method according to p. 16, in which the specified stage of introducing includes introducing the specified related to the mammalian recipient molecule is a nucleic acid that encodes a specified Oligopeptide, and specified Oligopeptide is expressed by the specified nucleic acid molecule.

19. The method according to p. 17 or 18, where this nucleic acid molecule is contained in the virus.

20. The method of inhibition of protein synthesis of inflammatory cytokines by cells capable of producing the specified protein inflammatory cytokine, and the method includes connecting these cells with the overwhelming synthesis of inflammatory cytokines by the number of connections, including Oligopeptide according to any one of paragraphs.1-6; and the synthesis of the specified inflammatory cytokine is suppressed.

21. The method according to p. 20, in which the specified stage of connection is carried out in the presence of a viable solid organ or viable cells, kotorom this protein inflammatory cytokine selected from the group consisting of factor-necrosis of tumor cells,-interferon, IL-1, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, 1L-16 and MlPl.
23. The method according to p. 20, in which the specified stage connection includes providing contact of these cells with a nucleic acid molecule that encodes a specified Oligopeptide; and specified Oligopeptide is expressed by the specified nucleic acid molecule.

24. The method according to p. 20, where this nucleic acid molecule is contained in the virus.

25. The method of suppressing the inflammatory response in mammals, and this method includes providing contact specified mammal with an overwhelming inflammatory response number of compounds, including Oligopeptide according to any one of paragraphs.1-6; and specified inflammatory response is suppressed.

26. The method according to p. 25, in which the specified stage of implementation of the contact includes an introduction to the specified mammal molecule is a nucleic acid that encodes a specified Oligopeptide, and specified Oligopeptide is expressed by the specified nucleic acid molecule.

27. The method according to p. 26, where this nucleic acid molecule is contained in virum arthritis, Crohn's disease or colitis.

29. The method of modulation of the activity hem-comprising enzyme, and this method includes providing contact system, including the specified enzyme, modulating the activity of a number of compounds, including Oligopeptide according to any one of paragraphs.1-6; moreover, the activity specified hem-comprising enzyme is modulated.

30. The method according to p. 29, wherein said hem-comprising an enzyme selected from the group consisting of hemoxygenase, nitric oxide synthase, cyclooxygenase, and guanylate cyclase.

31. The method according to p. 29, in which the specified stage of implementation of the contact includes an introduction to the specified system nucleic acid molecule that encodes a specified Oligopeptide; and specified Oligopeptide is expressed by the specified nucleic acid molecule.

32. The method according to p. 31, where this nucleic acid molecule is contained in the virus.

33. Way to delay the onset of autoimmune disease in a mammal at risk of developing a specified disease, and this method includes the introduction of the specified mammal overwhelming autoimmune disease number of compounds, including Oligopeptide according to any one of paragraphs.1-6; thus the offensive autoboy insulin-dependent diabetes mellitus, rheumatoid arthritis or systemic eritematoso lupus.

35. The method according to p. 33, in which the specified stage of introducing includes introducing the specified mammal molecule is a nucleic acid that encodes a specified Oligopeptide; and specified Oligopeptide is expressed by the specified nucleic acid molecule.

36. The method according to p. 35, where this nucleic acid molecule is contained in the virus.

 

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The invention relates to peptides consisting of 16-55 amino acid residues, the peptides comprise at least one of the amino acid sequence: FLCTHIIYS (SEQ ID NO:61), IIYSFANIS (SEQ ID NO: 62), FIKSVPPFL (SEQ ID NO:64), FDGLDLAWL (SEQ ID NO:65), LYPGRRDKQ (SEQ ID NO: 66), YDIAKISQH (SEQ ID NO:67), LDFISIMTY (SEQ ID NO:68), FISIMTYDF (SEQ ID NO: 69), FRGQEDASP (SEQ ID NO: 70), YAVGYMLRL (SEG ID NO:71), MLRLGAPAS (SEQ ID NO:72), LAYYEICDF (SEQ ID NO:73), LRGATVHRT (SEQ ID NO: 74), YLKDRQLAG (SEQ ID NO:75), LAGAMVWAL (SEQ ID NO:76), VWALDLDDF (SEQ ID NO: 77) or LDLDDFQGS (SEQ ID NO:78)

The invention relates to new peptides of General formula 1 (H-a-Lys-d-Trp-Lys-c - Pro-d-Lys-Pro-Trp-e-Arg-NH2where a = -Ile or Lys; b= -Pro - or-Lys-; C, d = -Lys or Trp; e=Arg or Ala, having biocidal activity, which combine higher compared to indolizidines biocidal activity with no damaging effects on blood cells

The invention relates to new peptide-based molecules interleukin-8 General formula

< / BR>
stimulating migration of neutrophils

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The invention relates to biotechnology
The invention relates to a group of new protected linear peptides containing the amino acid sequence, which can be used as starting compounds to obtain RGD-containing cyclopeptides, the General formula

R3-Arg-Gly-Asp(OR1)-OR2,

where R1is benzyl or tert-butyl; R2not equal to R1and is selected from the group of tert-butyl; benzyl; 4-methoxybenzyl; 4-nitrobenzyl; diphenylmethyl; 2,2,2-trichloroethyl; 2,2,2-trichloro-1,1-dimethylethyl; allyl; 9-fluorenylmethyl; carboxamidine; substituted 2-sulfonylated type AND-SO2-CH2-CH2- where a is substituted or unsubstituted phenyl or benzyl; R3is a hydrogen atom or a urethane protective group of the B1O-CO-, where1not equal to R1and can take values tert-butyl, benzyl, 4-methoxybenzyl, 9-fluorenylmethyl, 2-(4-nitrophenyloctyl)ethyl; or is a peptidyl containing from one to three amino acid residues; and the peptides, where R3- peptidyl structure E-Z-Y-X-, in which E is a hydrogen atom or a urethane protective group IN2O-CO-, where2not equal to R1and can take values tert-butyl; benzyl; 4-methoxybenzyl; 9-fluorenylmethyl; 2-(4-nitrophen is IN3O-CO-, in which3= R1attached to the omega-amino group; Z can take values Phe or D-Phe

The invention relates to a new compound - peptide of General formula Thr-Gly-Glu-Asn-His-Arg, possessing the biological activity of inducing differentiation and inhibiting cell proliferation in tumors and activity of the tread and the normalizing action on the vital processes of mammalian cells obtained by the method of solid-phase peptide synthesis by sequential growth of the peptide chain and having the following properties: mol

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The invention relates to a group of new protected linear peptides containing the amino acid sequence, which can be used as starting compounds to obtain RGD-containing cyclopeptides, the General formula

R3-Arg-Gly-Asp(OR1)-OR2,

where R1is benzyl or tert-butyl; R2not equal to R1and is selected from the group of tert-butyl; benzyl; 4-methoxybenzyl; 4-nitrobenzyl; diphenylmethyl; 2,2,2-trichloroethyl; 2,2,2-trichloro-1,1-dimethylethyl; allyl; 9-fluorenylmethyl; carboxamidine; substituted 2-sulfonylated type AND-SO2-CH2-CH2- where a is substituted or unsubstituted phenyl or benzyl; R3is a hydrogen atom or a urethane protective group of the B1O-CO-, where1not equal to R1and can take values tert-butyl, benzyl, 4-methoxybenzyl, 9-fluorenylmethyl, 2-(4-nitrophenyloctyl)ethyl; or is a peptidyl containing from one to three amino acid residues; and the peptides, where R3- peptidyl structure E-Z-Y-X-, in which E is a hydrogen atom or a urethane protective group IN2O-CO-, where2not equal to R1and can take values tert-butyl; benzyl; 4-methoxybenzyl; 9-fluorenylmethyl; 2-(4-nitrophen is IN3O-CO-, in which3= R1attached to the omega-amino group; Z can take values Phe or D-Phe
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