Mycobacteria tuberculosis chaperonin 60.1 peptides and uses thereof

Abstract

To provide novel peptides derivable from the polypeptide chaperonin 60.1 and their use in a medicinal field, such as for the prevention and / or treatment of inflammatory conditions.SOLUTION: The present invention provides novel specific sub-fragments and variants of the sequence DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD that exhibits anti-inflammatory activity and is shown to significantly reduce the recruitment of eosinophils in an animal model of allergic airway inflammation, wherein they exhibit biological activity and an improved pharmacokinetic profile.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to novel peptides derived from the Mycobacterium tuberculosis polypeptide chaperonin 60.1 and their use in medicine, for example for the prevention and / or treatment of inflammatory diseases.

[0002] International Patent Application, Publication No. WO02 / 040037 discloses pharmaceutical compositions containing Mycobacterium tuberculosis Cpn60.1 (MtCpn60.1), as well as nucleic acid molecules encoding MtCpn60.1. The application also discloses several specific peptide fragments with similar biological activity that can be derived from the full-length polypeptide. Various therapeutic uses of these molecules are also disclosed, including the treatment and / or prevention of autoimmune diseases, allergic diseases, diseases characterized by a Th2-type immune response, and eosinophilia-related diseases.

[0003] International Patent Application, Publication No. WO2009 / 106819 discloses a series of novel peptides derived from MtCpn60.1, including a peptide designated "peptide 4" having the amino acid sequence: DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 1). SEQ ID NO: 1 has been shown to exhibit anti-inflammatory activity and to significantly reduce eosinophil recruitment in an animal model of allergic airway inflammation.

[0004] WO2013057499 discloses several small fragments of SEQ ID NO:1 that have been found to have biological activity. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 02 / 040037 Brochure [Patent Document 2] International Publication No. 2009 / 106819 Brochure [Patent Document 3] International Publication No. 2013 / 057499 Brochure Summary of the Invention

[0006] The present invention is based on the unexpected discovery that certain novel small fragments and variants of SEQ ID NO: 1 (DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD) exhibit biological activity and improved pharmacokinetic properties. The novel peptides of the present invention have relatively short amino acid chain lengths that favor high-yield preparation and isolation, making them particularly suitable for pharmaceutical development. Such peptides have also been shown to have improved in vivo biological stability compared to MtCpn60.1 and known peptide fragments thereof. More specifically, the peptides claimed herein have improved half-life.

[0007] Thus, in one aspect of the present invention, the present application provides an isolated or recombinant peptide molecule, which consists of an amino acid sequence selected from one of groups (i) to (ix), or a functionally equivalent fragment or variant thereof: (i) the sequence of SEQ ID NO: 2, XHGLNVNTLSYGD (SEQ ID NO: 2), in which X is absent or is selected from the group consisting of a beta-alanine residue, 8-amino-3,6-dioxaoctanoic acid, and an acetyl group; or a variant thereof comprising one or more of i(i) to i(iii); (i) one or more amino acid residues are in D-form; i(ii) GLNVNTLSYGD (SEQ ID NO:26) is inverted; or i(iii) the carboxy-terminal amino acid residue has been converted to a primary carboxamide group; (ii) DGSVVVNKVSEL-NH2 (SEQ ID NO:3); (iii) SELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 4): (iv) SELPAGHGLNVNTLS (SEQ ID NO:5); (v) PAGHGLNVNTLS-NH2 (SEQ ID NO:6); (vi) VVVNKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 7); (vii) NKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO:8); (viii) PAGHGLNVNTLSYGDLAAD (SEQ ID NO: 9); and (ix) HGLNVNTLSYGDLAAD (sequence number 10).

[0008] The inventors have surprisingly found that said peptides have a significantly improved plasma half-life compared to known peptides derived from said chaperonin 60.1, while at the same time being effective as anti-inflammatory drugs.

[0009] The peptides of the present invention are chemically or recombinantly synthesized and have several distinct chemical, structural, and functional properties from full-length chaperonin 60.1.

[0010] By "functionally equivalent" is meant any peptide and / or variant or fragment thereof having the same or substantially similar function (e.g. biological activity) as any function exhibited by or attributed to one or more of the defined amino acid sequences (i) to (ix). For example, a peptide consisting of the amino acid sequence defined in (i) to (ix) reduces the trafficking of eosinophils and / or neutrophils to sites of inflammation, and can therefore be used to prevent and / or treat various diseases and disorders, such as asthma, multiple sclerosis, Crohn's disease, rheumatoid arthritis, and inflammatory bowel disease. Functional equivalence with respect to a particular biological activity can be assessed using conventional models and methods; for example, by measuring inflammogen-induced eosinophil or neutrophil influx into the lungs in sensitized (inflammogen - ovalbumin) or naive (inflammogen - LPS) animals.

[0011] By "variant" is meant a peptide having an amino acid sequence with 66% or more identity to sequences (i)-(ix) above, e.g., 70%, 75%, 80%, 85%, 90%, or 95% or more. Thus, the term "variant" refers to polypeptides and peptides that differ from naturally occurring molecules by amino acid insertions, deletions, and substitutions, e.g., made using recombinant DNA technology. Guidance for determining which amino acid residues can be substituted, added, or deleted without eliminating the desired activity can be found by comparing the sequence of a particular polypeptide with that of a homologous peptide and minimizing the number of amino acid sequence changes that occur in regions of high homology (conserved regions) or by replacing amino acids with consensus sequences.

[0012] Alternatively, recombinant variants encoding these same or similar polypeptides can be synthesized or selected by exploiting the "redundancy" of the genetic code. Various codon substitutions, such as silent changes resulting in various restriction enzyme sites, can be introduced to optimize cloning into plasmid or viral vectors or expression in specific prokaryotic or eukaryotic cell systems. Mutations in the polynucleotide are reflected in the polypeptide, but domains of other peptides can also be added to the polypeptide to modify the properties of any part of the polypeptide, changing properties such as ligand binding affinity, interchain affinity, or degradation / turnover rate.

[0013] Preferably, an amino acid "substitution" is the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, i.e., a conservative amino acid substitution. "Conservative" amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include aspartic acid and glutamic acid. "Insertions" and "deletions" preferably range from about 1-10 amino acids, but more preferably 1-5 amino acids, e.g., 1, 2, 3, 4, or 5 amino acids. Tolerable mutations can be identified experimentally by using recombinant DNA techniques to systematically insert, delete, or substitute amino acids into a polypeptide molecule and assaying the biological activity of the resulting recombinant variants.

[0014] Alternatively, where altered function is desired, insertions, deletions, or non-conservative mutations can be designed to generate modified polypeptides. Such mutations can, for example, change one or more biological functions or biochemical properties of the polypeptides of the invention. For example, such mutations can alter properties of the polypeptide, such as ligand binding affinity, interchain affinity, or degradation / turnover rate. Furthermore, such mutations can be selected to generate polypeptides that are better suited for expression, scale-up, etc. in the host cell selected for expression.

[0015] Fragments of the peptides of the present invention that can exhibit biological activity are also included in the present invention. Such fragments may be linear, but may also be cyclized using known methods, for example, as described in HU Saragovi, et al., Bio / Technology 10, 773-778 (1992) and RS McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference. Such fragments may also be fused to carrier molecules, such as immunoglobulins, for various applications, including increasing the valency of protein binding sites.

[0016] "Identity" means the number or percentage (depending on the presentation of the results) of amino acid residues in a candidate sequence that are identical with the amino acid residues in the sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity; conservative substitutions are not considered part of sequence identity.

[0017] The percentage sequence identity between two polypeptides can be determined using a suitable computer program, such as, for example, the GAP program of the University of Wisconsin Genetic Computing Group, although it will be appreciated that the percentage identity is calculated for polypeptides with optimally aligned sequences. The alignment can also be performed using the Clustal W program (Thompson et al., (1994) Nucleic Acids Res. 22, 4673-80). The parameters used can be as follows: Fast Pairwise Alignment parameters: K-tuple (word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent; Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05. Scoring matrix: BLOSUM.

[0018] Sequence identity can be determined, for example, using the Jotun Hein method (Hein, J. (1990) Methods Enzymol. 183:626-645). Identity between sequences can also be determined by other methods known in the art, for example, by varying hybridization conditions.

[0019] A preferred embodiment of the present invention relates to an isolated or recombinant peptide molecule consisting of an amino acid sequence selected from one of groups (a) to (n): (a) XHGLNVNTLSYGD-NH2 (SEQ ID NO: 11); (b) XdGysltnvnlGh-NH2 (SEQ ID NO: 12); (c) XhGlnvntlsyGd-NH2 (SEQ ID NO: 13); (d) hGLNVNTLSYGd-NH2 (SEQ ID NO: 14); (e) HGLNVNTLSYGd-NH2 (SEQ ID NO: 15); (f) hGLNVNTLSYGD-NH2 (SEQ ID NO: 16); (g) DGSVVVNKVSEL-NH2 (SEQ ID NO:3); (h) SELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 4); (i) SELPAGHGLNVNTLS (SEQ ID NO:5); (j) PAGHGLNVNTLS-NH2 (SEQ ID NO:6); (k) VVVNKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 7); (l) NKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO:8); (m) PAGHGLNVNTLSYGDLAAD (SEQ ID NO: 9); and (n) HGLNVNTLSYGDLAAD (SEQ ID NO: 10); where capital letters indicate L-amino acid residues, lower case letters indicate D-amino acid residues, and X is absent or selected from the group consisting of β-alanine residue, 8-amino-3,6-dioxaoctanoic acid, and an acetyl group.

[0020] The nomenclature of amino acids and peptide derivatives follows the IUPAC-IUB rules (J. Peptide Sci. 1999, 5, 465-471). D-amino acids are represented by lower case abbreviations, e.g., L-alanine is represented by Ala or A, and D-alanine is represented by ala or a.

[0021] The peptide of the present invention preferably consists of 5 to 40 amino acid residues, and more preferably consists of 5 to 30, or 5 to 20 amino acid residues.

[0022] In a preferred embodiment, the isolated or recombinant peptide molecule consists of the amino acid sequence HGLNVNTLSYGD-NH2 (SEQ ID NO: 17), or a functionally equivalent fragment or variant thereof.

[0023] In a preferred embodiment, the isolated or recombinant peptide molecule consists of the amino acid sequence bAla-HGLNVNTLSYGD-NH2 (SEQ ID NO: 18), or a functionally equivalent fragment or variant thereof.

[0024] In another preferred embodiment, the isolated or recombinant peptide molecule consists of the amino acid sequence Ac-dGysltnvnlGh-NH2 (SEQ ID NO: 19), Ac-hGlnvntlsyGd-NH2 (SEQ ID NO: 20), or a functionally equivalent fragment or variant thereof.

[0025] In another preferred embodiment, the present invention provides an isolated or recombinant peptide molecule consisting of the amino acid sequence hGLNVNTLSYGd-NH2 (SEQ ID NO: 14), or a functionally equivalent fragment or variant thereof.

[0026] In yet another preferred embodiment, the present invention provides an isolated or recombinant peptide molecule consisting of the amino acid sequence HGLNVNTLSYGd-NH2 (SEQ ID NO: 15); or a functionally equivalent fragment or variant thereof.

[0027] In yet another preferred embodiment, there is provided an isolated or recombinant peptide molecule consisting of the amino acid sequence hGLNVNTLSYGD-NH2 (SEQ ID NO: 16); or a functionally equivalent fragment or variant thereof.

[0028] In another preferred embodiment, there is provided an isolated or recombinant peptide molecule consisting of the amino acid sequence DGSVVVNKVSEL-NH2 (SEQ ID NO:3); or a functionally equivalent fragment or variant thereof.

[0029] MtCpn60.1 can be cloned and expressed using the methods described in TH Kong et al., Proc. Natl. Acad. Sci., 1993, 90, 2608-2612, and JC Lewthwaite et al, Infection and Immunity, 2001, 69(12), 7349-7355. MtCpn60.1 is also commercially available from Lionex (Germany).

[0030] The peptides of the present invention can be prepared and / or isolated using conventional methods known in the art. For example, by conventional liquid or solid phase synthesis or using an automated solid phase synthesiser, for example, as described in I. Coin, Nature Protocols, 2007, 2, 3247-3256. Preferably, the peptides of the present invention are prepared by Fmoc solid phase synthesis using a method similar to that described in GB Fields and RL Noble, Int. J. Peptide Protein Res., 1990, 35(3), 161-214. The peptides can be cyclized by known methods, for example, as described in HU Saragovi, et al., Bio / Technology 10, 773-778 (1992), and RS McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference. Such peptides can be fused to carrier molecules, such as immunoglobulins, for many purposes, including increasing the valency of protein binding sites.

[0031] It is understood that the compounds of the present invention include salts. Metabolites and prodrugs are also included. Compounds according to the present invention also include any isotopic derivatives.

[0032] The peptides can be used in accordance with the present invention when the patient is receiving one or more other therapeutic agents or when the peptides are given in combination with one or more therapeutic agents. The therapeutic agents can be selected from, but are not limited to, disease-modifying drugs such as bioimmunomodulators, analgesics, bronchodilators, anti-inflammatory drugs, anti-allergy drugs, allergen immunotherapy drugs, antiviral drugs, antibiotics, antibodies, steroids, and drugs commonly used to treat any of the chronic relapsing-remitting diseases in accordance with the present invention.

[0033] Disease-modifying drugs include, for example, hydroxychloroquine, sulfasalazine, leflunomide, methotrexate, and minocycline, as well as biologics targeting TNFα, such as abatacept, adalimumab, etanercept, infliximab, and golimumab, or immunomodulatory drugs, such as alemtuzumab, interferon beta-1b, beta interferon-1a, dimethyl fumarate, copaxone, natalizumab, and teriflunomide. Analgesics include paracetamol, nonsteroidal anti-inflammatory drugs, such as ibuprofen and aspirin, codeine, tramadol, morphine, amitriptyline, gabapentin, and opiates.

[0034] Anti-inflammatory drugs include leukotriene receptor antagonists, theophylline and PDE4 inhibitors, such as roflumilast, low-, medium- and high-dose corticosteroids for inhalation, subcutaneous, intramuscular, sublingual, intravenous and oral administration. Antiviral drugs include oseltamivir. Antibiotics include amoxicillin. Antibodies include anti-IgE antibodies (e.g. omalizumab), antibodies that modify cytokine signaling (e.g. anti-IL-5 monoclonal antibody mepolizumab). Steroids include fluticasone propionate and fluticasone furoate, beclomethasone dipropionate, budesonide, ciclesonide, flunisolide and mometasone. The use of the present invention may be preferred if the additional therapeutic agent or agents are selected from corticosteroids, anti-leukotrienes, cytokine monoclonal antibodies, or theophylline. The use may also be preferred if the additional agent is a bronchodilator. Preferred bronchodilators include short-acting beta2 agonists, such as salbutamol, long-acting beta2 agonists, such as salmeterol, formoterol, olodaterol and vilanterol, short-acting muscarinic receptor antagonists, such as ipratropium bromide, and long-acting muscarinic receptor antagonists, such as aclidinium bromide, tiotropium bromide, and glycopyrronium bromide.

[0035] Use of peptides According to a further aspect of the present invention there is provided a peptide as described herein for use in human or veterinary medicine.

[0036] The peptides of the invention may be for use in humans or non-human animals, typically mammals.

[0037] Preferably, the peptide of the present invention is used for treating and / or preventing acute and / or chronic inflammatory disease.Preferable examples of acute and / or chronic inflammatory disease include inflammation associated with infection (such as septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury such as coronary artery thrombosis and cerebral artery occlusion, shock lung syndrome, endotoxin lethality, arthritis (particularly rheumatoid arthritis or chronic inflammatory arthritis), complement-associated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, non-allergic asthma, allergic asthma, allergic rhinitis, atopic dermatitis, cystic fibrosis, pulmonary fibrosis, inflammatory bowel disease, Crohn's disease, and ulcerative colitis.

[0038] Other preferred inflammatory conditions include: acute organ failure associated with surgery, such as the following surgical interventions (not an exhaustive list): cardiac surgery, coronary artery bypass, aortic aneurysm, valve surgery, esophagectomy, kidney disease, coronary artery bypass graft surgery, aortic root and ascending aortic aneurysm repair surgery, acute kidney injury, acute respiratory distress syndrome, and the like.

[0039] In another embodiment, the present invention provides the use of a peptide as described herein for the prevention and / or treatment of chronic obstructive pulmonary disease.

[0040] In yet another embodiment, the present invention provides the use of the peptides described herein for the prevention and / or treatment of autoimmune diseases.

[0041] As used herein, the term "autoimmune disease" includes diseases in which an autoimmune process may be demonstrated to contribute to the pathogenesis of the disease. Such diseases are typically associated with a helper T-lymphocyte-1 (Th-1) type immune response.

[0042] Examples of autoimmune diseases that can be prevented and / or treated by the peptide molecules of the present invention include autoimmune diseases such as hemolytic anemia, thrombocytopenia, pernicious anemia, Addison's disease, autoimmune diabetes, insulin-dependent diabetes mellitus, myasthenia gravis, rheumatoid arthritis, systemic lupus erythematosus, atherosclerosis, autoimmune encephalitis, connective tissue disease, multiple sclerosis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, graft-versus-host disease, and autoimmune inflammatory eye disease. Preferred autoimmune diseases include rheumatoid arthritis and multiple sclerosis.

[0043] In another embodiment, the present invention provides the use of the peptides described herein for the prevention and / or treatment of allergic diseases. Examples of allergic diseases that can be prevented and / or treated by the peptide molecules of the invention include eczema, dermatitis, allergic rhinitis (hay fever), allergic airway disease, hypereosinophilic syndrome, contact dermatitis; respiratory diseases such as asthma, including allergic asthma and intrinsic asthma, characterized by eosinophilic airway inflammation and airway hyperresponsiveness, allergic bronchopulmonary aspergillosis, eosinophilic pneumonia, allergic bronchitis bronchiectasis, occupational asthma, airway hyperresponsiveness syndrome, interstitial lung disease, hypereosinophilic syndrome, pulmonary infestation; anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, and giant papillary conjunctivitis. Other diseases in which immune modulation is desirable (including, for example, organ transplantation) may also be treated using the peptide molecules of the invention. Preferred allergic diseases include asthma, allergic rhinitis, and atopic dermatitis.

[0044] As used herein, the terms "allergic disease" and "allergic condition" include conditions associated with a helper T lymphocyte-2 (Th-2) type immune response. In an allergic response, high IgE levels occur, resulting in a dominance of the Th-2 response over the Th-1 response, resulting in an inflammatory response.

[0045] WO2013057499 discloses suitable assays for evaluation; (a) the therapeutic effect of the polypeptide or its antagonist on an allergic response; (b) Thymocyte or splenocyte cytotoxicity (c) T cell-dependent immunoglobulin responses and isotype switching, which identifies, among other things, proteins that modify T cell-dependent antibody responses and affect the Th1 / Th2 profile; (d) Mixed Lymphocyte Reaction (MLR) Assays, which specifically identify proteins that generate primarily Th1 and CTL responses. (e) Dendritic cell-dependent assays, which specifically identify proteins expressed by dendritic cells that activate naive T cells. (f) lymphocyte survival / apoptosis (this specifically identifies proteins that prevent apoptosis following superantigen induction and proteins that control lymphocyte homeostasis); (g) proteins that affect early stages of T cell commitment and development. (h) The immunomodulatory effects of the peptide molecules of the invention on rheumatoid arthritis can be determined in experimental animal model systems.

[0046] The term "treatment" as used herein means the reduction, alleviation, or elimination of one or more symptoms of the disease being treated compared to the symptoms prior to treatment. For example, symptoms that may be affected include eosinophilia, decreased secretion of certain cytokines, Th2-based immune responses, allergic responses, and the presence of autoantibodies.

[0047] The term "prophylaxis" as used herein means delaying or preventing the onset of a disease or reducing its severity as assessed by the appearance or extent of one or more symptoms of said disease.

[0048] The present invention further provides a method for preventing and / or treating the above diseases, comprising administering to a mammal, including a human, a peptide as described herein.

[0049] The present invention further provides the use of a peptide as described herein in the manufacture of a medicament for the prevention and / or treatment of the above diseases.

[0050] Pharmaceutical Compositions In yet another aspect, the present invention provides a pharmaceutical composition comprising the above peptide molecule and one or more pharma- ceutically acceptable excipients for use in the above methods.

[0051] The compounds described herein can be formulated for administration in any convenient manner. The present invention provides pharmaceutical compositions comprising the peptide molecules described above and one or more pharma- ceutically acceptable excipients for use in the above methods.

[0052] Any suitable route of administration may be used, for example oral, topical, parenteral, ocular, rectal, vaginal, inhalation, buccal, sublingual, and intranasal routes of administration are all contemplated as suitable.

[0053] The peptide molecules and pharmaceutical compositions of the present invention can be administered parenterally, for example, intravenously, intraarterially, intraperitoneally, intrathecally, intracerebroventricularly, intracisternally, intracranially, intramuscularly, or subcutaneously, but may also be administered by infusion techniques. They are best used in the form of a sterile aqueous solution, which may contain other substances, for example, sufficient salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. Preparation of suitable parenteral formulations under sterile conditions is accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0054] Drugs and pharmaceutical compositions suitable for parenteral administration include sterile aqueous and non-aqueous injection solutions containing antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and sterile aqueous and non-aqueous suspensions containing suspending and thickening agents. Drugs and compositions may be provided in single-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a lyophilized state, requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets, as previously described.

[0055] The molecules, agents and pharmaceutical compositions of the present invention can also be administered intranasally or by inhalation, conveniently in the form of a dry powder inhaler or in the form of an aerosol spray from a pressurized container, pump, spray or nebulizer, using as a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, hydrofluoroalkanes such as 1,1,1,2-tetrafluoroethane (HFA 134A3) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be measured by providing a valve to administer a metered amount. The pressurized container, pump, spray or nebulizer contains a solution or suspension of the active drug, for example, in a mixture of ethanol and propellant as a solvent, and may additionally contain a lubricant such as sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler may be formulated to contain a powder mix of a drug of the invention and a suitable powder base such as lactose or starch.

[0056] Aerosol or dry powder formulations are preferably arranged so that each metered dose or "puff" contains at least 200 pg of a molecule of the invention for administration to a patient. Of course, the total daily dose of an aerosol will vary from patient to patient and may be administered in a single dose, but is more commonly administered in divided doses throughout the day.

[0057] The peptide molecules and pharmaceutical compositions of the present invention can also be administered orally. The process uses the natural process for oral intake of vitamin B12 and / or vitamin D in the body, and can deliver proteins and peptides simultaneously. By riding on the vitamin B12 and / or vitamin D uptake system, the nucleic acids, molecules and pharmaceutical compositions of the present invention can pass through the intestinal wall. A complex is synthesized between vitamin B12 analogues and / or vitamin D analogues and drugs, which complex retains both the significant affinity for intrinsic factor (IF) in the vitamin B12 / vitamin D part of the complex, and the significant biological activity of the active substance of the complex.

[0058] The peptide molecules and pharmaceutical compositions of the present invention are administered in the form of a pharmaceutical composition containing the active ingredient, usually by any parenteral route or intranasally, but in some embodiments orally. Depending on the disease and patient to be treated, and the route of administration, the compositions are administered in various doses.

[0059] In human therapy, the peptide molecules and pharmaceutical compositions of the invention may be administered alone, but will most often be administered in admixture with a suitable excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

[0060] Preferably, the pharmaceutical compositions of the present invention are unitary dosage forms containing a daily dose or unit amount, daily sub-dose, or an appropriate fraction thereof, of the active ingredient.

[0061] For example, the peptide molecules and pharmaceutical compositions of the present invention can be administered orally, bucally, or sublingually in the form of tablets, capsules, suppositories, elixirs, solutions or suspensions (which may contain flavoring or coloring agents) for immediate, delayed, or controlled release applications. The peptide molecules and pharmaceutical compositions of the present invention can also be administered by intracavernosal injection.

[0062] Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, calcium hydrogen phosphate and glycine, disintegrants such as starch (corn, potato, or tapioca starch), sodium starch glycolate, croscarmellose sodium and complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and gum arabic. Additionally, lubricants such as magnesium stearate, stearic acid, and glyceryl behenate may be included.

[0063] Similar solid compositions may be used as excipients for gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and / or elixirs, the drugs of the present invention may be mixed with various sweetening or flavoring agents, coloring substances or dyes, mixed with emulsifying and / or suspending agents, and mixed with diluents such as water, ethanol, propylene glycol, and glycerin, as well as combinations thereof.

[0064] Daily dosage levels of the molecules, agents and pharmaceutical compositions of the invention for oral and parenteral administration to human patients will typically be in the range of 200 pg to 100 mg per adult per day, administered in single or divided doses.

[0065] Thus, for example, a vial, tablet or capsule of a molecule of the invention may contain from 200 pg to 100 mg of active agent for administration one at a time, or two at a time, or three or more at a time, as appropriate. The physician will in any event determine the exact dosage that will be most suitable for an individual patient, which will vary with the age, weight and response of the particular patient. The dosages given above are exemplary of the average case. There may, of course, be unique instances in which higher or lower dosage ranges are merited, and such are within the scope of the present invention.

[0066] Alternatively, the molecules, agents and pharmaceutical compositions of the present invention may be administered as a suppository or pessary, but may also be applied topically as a lotion, liquid, cream, gel, ointment, or powder. The molecules, agents and pharmaceutical compositions of the present invention may also be administered transdermally, for example, by using a skin patch. They may also be administered intraocularly, particularly to treat eye diseases.

[0067] For ophthalmic use, the molecules, agents and pharmaceutical compositions of the invention can be formulated as micronized suspensions in sterile, pH-adjusted, isotonic saline, or, preferably, as solutions in sterile, pH-adjusted, isotonic saline, optionally combined with a preservative such as benzalkonium chloride, or alternatively, in an ointment such as petrolatum.

[0068] For topical application to the skin, the molecules, agents and pharmaceutical compositions of the invention can be formulated as a suitable ointment containing the active drug suspended or dissolved in one or more of the following, for example: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene agent, emulsifying wax, and water, or as a suitable lotion or cream suspended or dissolved in one or more of the following, for example: mineral oil, sorbitan monostearate, polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0069] Formulations suitable for topical administration in the mouth include lozenges containing the active ingredient in a flavored base, usually sucrose and acacia or tragacanth; lozenges containing the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes containing the active ingredient in a suitable liquid base.

[0070] For veterinary use, the molecules, medicaments and pharmaceutical compositions of the invention will be administered in any appropriately accepted formulation in accordance with normal veterinary practice, with the veterinarian being able to determine the dosage regimen and route of administration which will be most suitable for the individual animal.

[0071] The formulation is advantageously a pharmaceutical formulation. The formulation is advantageously a veterinary formulation.

[0072] Advantageously, according to the present invention, the daily dosage level is between 10 pg and 100 mg. Preferably, the daily dosage level is between 20 pg and 50 mg, between 20 pg and 10 mg, or between 20 pg and 8 mg, administered in a single dose or in divided doses.

[0073] Preferred pharmaceutical formulations include those in which the active ingredient is present at 0.000001% to 5% by weight, in other words, the ratio of active ingredient to other components of the pharmaceutical composition (i.e., adjuvants, diluents and carriers) is at least 1:99 by weight (e.g., at least 10:90, preferably at least 30:70, and most preferably at least 50:50).

[0074] Preferably, the pharmaceutical composition or medicament of the present invention is formulated to allow administration by at least one route selected from the group consisting of or comprising: intranasal; oral; parenteral; topical; ophthalmic; suppository; vaginal suppository; or inhalation.Formulations suitable for such administration routes are well known to those skilled in the art of pharmacy and medicine, and typical formulations are described above and in the accompanying examples.

[0075] Typically, the patient is administered between 6 and 12 hours; in a preferred embodiment, the patient is administered between 9 and 12 hours after the previous administration; more preferably, the patient is administered between 12 hours and 12 days; even more preferably, the patient is administered between 12 days and 6 months.

[0076] The pharmaceutical composition or medicament of the present invention is preferably formulated to allow administration by at least one route, which route includes or is selected from the group consisting of intranasal; sublingual, oral; parenteral; topical; ophthalmic; suppository; vaginal suppository; or inhalation.Formulations suitable for such administration routes are well known to those skilled in the art of pharmacy and medicine, and typical formulations are described above and in the accompanying examples.

[0077] Description of the drawings Non-limiting examples are now described with reference to the following drawings: [Brief description of the drawings]

[0078] [Figure 1]FIG. 1 shows the effect of SEQ ID NO:1 (0.5 pg / mouse iv) on LPS-induced pulmonary neutrophil influx in mice when SEQ ID NO:1 is administered 0, 30 and 60 minutes prior to LPS challenge. [Diagram 2] FIG. 2 shows the effect of SEQ ID NO:3 (50 pg / mouse iv) on LPS-induced pulmonary neutrophil influx in mice when SEQ ID NO:3 is administered 0, 30, and 60 minutes prior to LPS challenge. [Diagram 3] FIG. 3 shows the effect of SEQ ID NO:3 (50 pg / mouse iv) on LPS-induced pulmonary neutrophil influx in mice when SEQ ID NO:3 is administered 0, 30, 60, and 120 minutes after LPS administration. [Figure 4] FIG. 4 shows the effect of SEQ ID NO:1 (50 pg / mouse iv) on LPS-induced pulmonary neutrophil influx in mice when SEQ ID NO:1 is administered 0, 30, 60 and 120 minutes after LPS administration. [Diagram 5] FIG. 5 shows the effect of SEQ ID NO: 17 (0.05, 0.5, and 5 pg / dose) on LPS-induced neutrophil influx into mouse lungs. [Figure 6] FIG. 6 shows the effect of SEQ ID NOs: 21, 22, and 18 (50 pg / mouse) on LPS-induced neutrophil influx into mouse lungs. [Figure 7] Figure 7 shows the effect of SEQ ID NO: 4 (0.5, 5 and 500 pg / dose) on LPS-induced neutrophil influx into mouse lungs. Example 1

[0079] synthesis Synthesis and purification were performed using automated fluorenylmethyloxycarbonyl solid-phase peptide synthesis (Fmoc SPSS). Peptides were synthesized using Fmoc / t-butyl solid-phase synthesis on 2-chlorotrityl or TentaGel resins derivatized with one of a number of cleavable linkers. Temporary N-amino group protection was provided by the Fmoc group, t-butyl ether was used for protection of the hydroxyl side chains of tyrosine, serine, and threonine, while t-butyl esters protected the side chains of aspartic and glutamic acid residues. Histidine and lysine side chains were protected as N-trityl and N-Boc derivatives, respectively, cysteine ​​as the S-trityl derivative, and the guanidine moiety of arginine as the Pbf derivative.

[0080] Upon completion of the synthesis, the peptides were cleaved from the solid support and the side chain protecting groups were removed by treatment with trifluoroacetic acid (TEA) containing triisobutylsilane and water as scavengers. After removing the TFA and scavengers by evaporation and trituration in diethyl ether, the peptides were purified by preparative reversed-phase HPLC and then lyophilized. The purified products were then analyzed by reversed-phase HPLC and mass spectrometry. Example 2

[0081] Activity of novel peptides in LPS inflammation models SEQ ID NO: 3 is a peptide according to the invention that is a fragment of the peptide of SEQ ID NO: 1 (Table 1) (peptide 4 described in WO2009 / 106819).

[0082] [Table 1]

[0083] LPS mouse method Female and male BALB / c mice, 6–8 weeks of age (Harlan, UK), were intranasally challenged with 25 μg / mouse Escherichia coli lipopolysaccharide (LPS) immediately after intranasal challenge with peptides diluted in sterile saline or saline alone. Four hours later, mice were euthanized with an overdose of urethane, and the exposed trachea was cannulated and the lungs were instilled with 0.5 ml saline three times. An aliquot (50 μl) of the BAL (bronchoalveolar lavage) fluid was added to 50 μl of hemolysis solution (Turk's solution, Fluka, UK). The total number of cells in the lavage fluid was counted with a modified Neubauer hemocytometer. To determine the white blood cell percentage, cytospin preparations were prepared from BAL fluid (100 μl) centrifuged at 1000 rpm for 1 min at room temperature. Cells were stained with Diff Quick and a total of 100 cells were counted to determine the percentage of neutrophils, eosinophils, and monocytes using standard morphological criteria.

[0084] SEQ ID NO:1 is highly potent in the mouse LPS model when administered intravenously immediately prior to LPS challenge, showing significant inhibition of LPS-induced pulmonary neutrophil influx, however, when given at earlier (Figure 1) or later (Figure 4) time points, SEQ ID NO:1 loses its activity in this model.

[0085] SEQ ID NO:3, a fragment of SEQ ID NO:1, is effective in the LPS mouse model when administered intravenously. Surprisingly, SEQ ID NO:3 is also effective when administered up to 60 minutes before (FIG. 2) and up to 120 minutes after (FIG. 3) LPS challenge in this model for LPS-induced pulmonary neutrophil influx, unlike SEQ ID NO:1 (FIGS. 1 and 4), which only shows efficacy when administered immediately before LPS challenge.

[0086] conclusion The expanded dosing options for SEQ ID NO:3 allow for clinical adoption of both therapeutic and prophylactic dosing protocols, a property that expands the number of indications that can be treated with this peptide. Example 3

[0087] Small fragments of SEQ ID NO:1 exhibit significant improvement in plasma stability Many peptides have low plasma stability due to metabolism by proteases. A small peptide fragment of SEQ ID NO:1 (peptide 4 in WO2009 / 106819) was synthesized and its efficacy in the LPS mouse lung neutrophilia model, as well as its plasma stability in mouse, rat, and human plasma (SEQ ID NO:17) were examined. This peptide was then modified and the resulting molecule was examined in the same system.

[0088] [Table 2]

[0089] Plasma stability in mouse, rat, and human plasma at 37°C Plasma: Mouse (C57 Black 6 male), rat (Sprague Dawley), and human, containing heparin as an anticoagulant.

[0090] Duplicate plasma samples were incubated with 3 μM peptide in a volume of 600 μl at 37°C with shaking. 50 μl aliquots were withdrawn after 2, 5, 30, 60, and 120 min and quenched into 150 μl ice-cold methanol containing an internal standard (tolbutamide). Samples were stored at -20°C for >2 h to allow protein precipitation. Samples were then centrifuged at 2500 x g and the resulting supernatants were transferred to 96-well plates for LC-MS / MS. T half was calculated for each peptide in all matrices.

[0091] result [Table 3] Addition of a β-alanine group to the N-terminus (SEQ ID NO: 18), C-terminus (SEQ ID NO: 21) or both termini (SEQ ID NO: 22) improved the stability of the peptide in plasma.

[0092] 5, 6 and 7 relate to Example 2 and show the results of peptides tested in the LPS mouse method described above.

[0093] Figures 5 and 6 show the effect of SEQ ID NO: 17 on LPS-induced pulmonary neutrophil influx in mice, where administration of various doses of SEQ ID NO: 17 (0.5, 5 or 50 pg / mouse) reduces neutrophil numbers.

[0094] FIG. 7 shows the effect of SEQ ID NOs: 21, 22, and 18 (50 pg / mouse, iv) on LPS-induced pulmonary neutrophil influx in mice.

[0095] SEQ ID NO: 18 retains anti-inflammatory activity in the LPS mouse model while showing a significant improvement in plasma half-life (Th) in mouse, rat and human plasma compared to the parent peptide SEQ ID NO: 17 (Table 3). SEQ ID NOs: 21 and 22 show better Th than SEQ ID NO: 17 but lose efficacy in the LPS mouse lung inflammation model. Thus, only the addition of a β-alanine group to the C-terminus improves plasma stability while retaining efficacy. Example 4

[0096] Table 4 shows the plasma stability data of SEQ ID NO:23 and SEQ ID NO:5 (peptide fragments 2 and 3 of WO2013057499) in three species.

[0097] [Table 4]

[0098] A longer fragment (SEQ ID NO: 4), an extended version of fragment 3, was synthesized and tested for plasma stability, and showed superior half-life (Table 5) and maintained in vivo efficacy in a mouse LPS pulmonary inflammation model (Figure 7).

[0099] [Table 5] Example 5

[0100] Plasma stability of modified peptides Structural modifications were then made to the peptide of SEQ ID NO: 17 to improve its plasma stability, as shown in Table 6. The plasma stability of the peptide was then evaluated under the same conditions as above.

[0101] [Table 6]

[0102] The plasma half-lives of the above peptides in mice, rats, and humans are shown in Table 7.

[0103] [Table 7]

[0104] Addition of a β-alanine group to one end of SEQ ID NO:17 significantly improved plasma stability while maintaining activity, whereas addition of this group to both ends improved stability even further (see SEQ ID NO:22) but resulted in abolishment of biological activity. β-alanine addition is known to increase the ability of synthetic protein fragments to withstand the degradative action of aminopeptidases and serum proteases.

[0105] Reconstitution of the fragments with amino acids in the d-configuration, which are rarely found in vivo (SEQ ID NOs: 19 and 20), shows increased stability in plasma, likely due to increased protection from proteolysis by aminopeptidases and serum proteases. Addition of N-terminal acetylation to these peptides, which is known to have a stabilizing effect, further enhanced the increased stability in plasma.

[0106] Surprisingly, cyclization (SEQ ID NO: 25), one of the most widely used methods to increase the conformational stability of peptides, did not improve stability in plasma.

[0107] [Sequence Listing] SEQUENCE LISTING <110> Revolo Biotherapeutics Limited <120> MYCOBACTERIA TUBERCULOSIS CHAPERONIN 60.1 PEPTIDES AND USES THEREOF <130> PA24-606 <140> <141> 2018-01-11 <150> GB 1700557.0 <151> 2017-01-12 <160> 26 <170> PatentIn version 3.5 <210> 1 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <400> 1 Asp Gly Ser Val Val Val Asn Lys Val Ser Glu Leu Pro Ala Gly His 1 5 10 15 Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp Leu Ala Ala Asp 20 25 30 <210> 2 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa is absent or is selected from the group consisting of a beta alanine residue, 9-amino-3,6-dioxaoctanoic acid, and an acetyl group <400> 2 Xaa His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp 1 5 10 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <220> <221> MISC_FEATURE <222> (12)..(12) <223> NH2 group <400> 3 Asp Gly Ser Val Val Val Asn Lys Val Ser Glu Leu 1 5 10 <210> 4 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <400> 4 Ser Glu Leu Pro Ala Gly His Gly Leu Asn Val Asn Thr Leu Ser Tyr 1 5 10 15 Gly Asp Leu Ala Ala Asp 20 <210> 5 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <400> 5 Ser Glu Leu Pro Ala Gly His Gly Leu Asn Val Asn Thr Leu Ser 1 5 10 15 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <220> <221> MISC_FEATURE <222> (12)..(12) <223> NH2 group <400> 6 Pro Ala Gly His Gly Leu Asn Val Asn Thr Leu Ser 1 5 10 <210> 7 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <400> 7 Val Val Val Asn Lys Val Ser Glu Leu Pro Ala Gly His Gly Leu Asn 1 5 10 15 Val Asn Thr Leu Ser Tyr Gly Asp Leu Ala Ala Asp 20 25 <210> 8 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <400> 8 Asn Lys Val Ser Glu Leu Pro Ala Gly His Gly Leu Asn Val Asn Thr 1 5 10 15 Leu Ser Tyr Gly Asp Leu Ala Ala Asp 20 25 <210> 9 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <400> 9 Pro Ala Gly His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp Leu 1 5 10 15 Ala Ala Asp <210> 10 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <400> 10 His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp Leu Ala Ala Asp 1 5 10 15 <210> 11 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa is absent or is selected from the group consisting of a beta alanine residue, 9-amino-3,6-dioxaoctanoic acid, and an acetyl group <220> <221> MISC_FEATURE <222> (13)..(13) <223> NH2 group <400> 11 Xaa His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp 1 5 10 <210> 12 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <220> <221> MISC_FEATURE <222> (1)..(1) <223> X is absent or is selected from the group consisting of a beta alanine residue, 9-amino-3,6-dioxaoctanoic acid, and an acetyl group <220> <221> MISC_FEATURE <222> (2)..(2) <223> D-amino acid residue <220> <221> MISC_FEATURE <222> (4)..(11) <223> D-amino acid residue <220> <221> MISC_FEATURE <222> (13)..(13) <223> D-amino acid residue <220> <221> MISC_FEATURE <222> (13)..(13) <223> NH2 group <400> 12 Xaa Asp Gly Tyr Ser Leu Thr Asn Val Asn Leu Gly His 1 5 10 <210> 13 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <220> <221> MISC_FEATURE <222> (1)..(1) <223> X is absent or is selected from the group consisting of a beta alanine residue, 9-amino-3,6-dioxaoctanoic acid, and an acetyl group <220> <221> MISC_FEATURE <222> (2)..(2) <223> D-amino acid residue <220> <221> MISC_FEATURE <222> (4)..(11) <223> D-amino acid residue <220> <221> MISC_FEATURE <222> (13)..(13) <223> D-amino acid residue <220> <221> MISC_FEATURE <222> (13)..(13) <223> NH2 group <400> 13 Xaa His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp 1 5 10 <210> 14 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <220> <221> MISC_FEATURE <222> (1)..(1) <223> D-amino acid residue <220> <221> MISC_FEATURE <222> (12)..(12) <223> D-amino acid residue <220> <221> MISC_FEATURE <222> (12)..(12) <223> NH2 group <400> 14 His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp 1 5 10 <210> 15 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <220> <221> MISC_FEATURE <222> (12)..(12) <223> D-amino acid residue <220> <221> MISC_FEATURE <222> (12)..(12) <223> NH2 group <400> 15 His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp 1 5 10 <210> 16 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <220> <221> MISC_FEATURE <222> (1)..(1) <223> D-amino acid residue <220> <221> MISC_FEATURE <222> (12)..(12) <223> NH2 group <400> 16 His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp 1 5 10 <210> 17 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <220> <221> MISC_FEATURE <222> (12)..(12) <223> NH2 group <400> 17 His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp 1 5 10 <210> 18 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <220> <221> MOD_RES <222> (1)..(1) <223> bAla <220> <221> MISC_FEATURE <222> (13)..(13) <223> NH2 group <400> 18 Xaa His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp 1 5 10 <210> 19 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <220> <221> MISC_FEATURE <222> (1)..(1) <223> Ac group <220> <221> MISC_FEATURE <222> (1)..(1) <223> D-amino acid residue <220> <221> MISC_FEATURE <222> (3)..(10) <223> D-amino acid residue <220> <221> MISC_FEATURE <222> (12)..(12) <223> D-amino acid residue <220> <221> MISC_FEATURE <222> (12)..(12) <223> NH2 group <400> 19 Asp Gly Tyr Ser Leu Thr Asn Val Asn Leu Gly His 1 5 10 <210> 20 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <220> <221> MISC_FEATURE <222> (1)..(1) <223> Ac group <220> <221> MISC_FEATURE <222> (1)..(1) <223> D-amino acid residue <220> <221> MISC_FEATURE <222> (3)..(10) <223> D-amino acid residue <220> <221> MISC_FEATURE <222> (12)..(12) <223> D-amino acid residue <220> <221> MISC_FEATURE <222> (12)..(12) <223> NH2 group <400> 20 His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp 1 5 10 <210> 21 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <220> <221> MOD_RES <222> (13)..(13) <223> bAla <220> <221> MISC_FEATURE <222> (13)..(13) <223> NH2 group <400> 21 His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp Xaa 1 5 10 <210> 22 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <220> <221> MOD_RES <222> (1)..(1) <223> bAla <220> <221> MOD_RES <222> (14)..(14) <223> bAla <220> <221> MISC_FEATURE <222> (14)..(14) <223> NH2 group <400> 22 Xaa His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp Xaa 1 5 10 <210> 23 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <400> 23 Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp Leu Ala Ala Asp 1 5 10 15 <210> 24 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <220> <221> MISC_FEATURE <222> (1)..(1) <223> O2Oc <220> <221> MISC_FEATURE <222> (12)..(12) <223> NH2 group <400> 24 His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp 1 5 10 <210> 25 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <220> <221> MISC_FEATURE <222> (1)..(12) <223> Cyclic peptide <400> 25 His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp 1 5 10 <210> 26 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence inverted for peptide molecule <400> 26 Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp 1 5 10

Claims

1. 1. An isolated or recombinant peptide molecule consisting of an amino acid sequence selected from one of the following groups (i) to (ix): (i) the sequence of SEQ ID NO: 2, XHGLNVNTLSYGD (SEQ ID NO: 2), in which X is absent or is selected from the group consisting of a beta-alanine residue, 8-amino-3,6-dioxaoctanoic acid, and an acetyl group; or a variant thereof comprising one or more of i(i) to i(iii); (i) one or more amino acid residues are in D-form; i(ii) GLNVNTLSYGD (SEQ ID NO:26) is inverted; or i(iii) the carboxy-terminal amino acid residue is converted to a primary carboxamide group; (ii) DGSVVVNKVSEL-NH2 (SEQ ID NO: 3); (iii) SELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 4): (iv) SELPAGHGLNVNTLS (SEQ ID NO:5); (v) PAGHGLNVNTLS-NH2 (SEQ ID NO: 6); (vi) VVVNKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 7); (vii) NKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 8); (viii) PAGHGLNVNTLSYGDLAAD (SEQ ID NO: 9); and (ix) HGLNVNTLSYGDLAAD (SEQ ID NO: 10) or a functionally equivalent fragment or variant thereof.

2. 2. The isolated or recombinant peptide molecule of claim 1, comprising an amino acid sequence selected from one of the following groups: (a) XHGLNVNTLSYGD-NH2 (SEQ ID NO: 11); (b) XdGysltnvnlGh-NH2 (SEQ ID NO: 12); (c) XhGlnvntlsyGd-NH2 (SEQ ID NO: 13); (d) hGLNVNTLSYGd-NH2 (SEQ ID NO: 14); (e) HGLNVNTLSYGd-NH2 (SEQ ID NO: 15); (f) hGLNVNTLSYGD-NH2 (SEQ ID NO: 16); (g) DGSVVVNKVSEL-NH2 (SEQ ID NO: 3); (h) SELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 4); (i) SELPAGHGLNVNTLS (SEQ ID NO:5); (j) PAGHGLNVNTLS-NH2 (SEQ ID NO: 6); (k) VVVNKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 7); (l) NKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 8); (m) PAGHGLNVNTLSYGDLAAD (SEQ ID NO: 9); and (n) HGLNVNTLSYGDLAAD (SEQ ID NO: 10); wherein capital letters represent L-amino acid residues, lower case letters represent D-amino acid residues, and X is absent or selected from the group consisting of a beta-alanine residue, 8-amino-3,6-dioxaoctanoic acid, and an acetyl group.

3. 3. The isolated or recombinant peptide molecule of claim 1 or 2, consisting of the amino acid sequence HGLNVNTLSYGD-NH2 (SEQ ID NO: 17), or a functionally equivalent fragment or variant thereof.

4. 3. The isolated or recombinant peptide molecule of claim 1 or 2, consisting of the amino acid sequence bAla-HGLNVNTLSYGD-NH2 (SEQ ID NO: 18), or a functionally equivalent fragment or variant thereof.

5. 3. The isolated or recombinant peptide molecule of claim 1 or 2, consisting of the amino acid sequence Ac-dGysltnvnlGh-NH2 (SEQ ID NO: 19), Ac-hGlnvntlsyGd-NH2 (SEQ ID NO: 20), or a functionally equivalent fragment or variant thereof.

6. 3. The isolated or recombinant peptide molecule of claim 1 or 2, consisting of the amino acid sequence hGLNVNTLSYGd-NH2 (SEQ ID NO: 14), or a functionally equivalent fragment or variant thereof.

7. 3. The isolated or recombinant peptide molecule of claim 1 or 2, consisting of the amino acid sequence HGLNVNTLSYGd-NH2 (SEQ ID NO: 15), or a functionally equivalent fragment or variant thereof.

8. 3. The isolated or recombinant peptide molecule of claim 1 or 2, consisting of the amino acid sequence hGLNVNTLSYGD-NH2 (SEQ ID NO: 16), or a functionally equivalent fragment or variant thereof.

9. 3. The isolated or recombinant peptide molecule of claim 1 or 2, consisting of the amino acid sequence DGSVVVNKVSEL-NH2 (SEQ ID NO: 3), or a functionally equivalent fragment or variant thereof.

10. 10. An isolated or recombinant peptide molecule according to any one of claims 1 to 9 for use in the medical and veterinary fields.

11. 11. The isolated or recombinant peptide molecule of claim 10 for use in the treatment and / or prevention of acute and / or chronic inflammatory diseases.

12. 12. The isolated or recombinant peptide molecule according to claim 11 for use according to claim 11, wherein the inflammatory disease is associated with eosinophilia and / or neutrophilia.

13. 13. The isolated or recombinant peptide molecule of claim 11 or 12 for use according to claim 11 or 12, wherein the inflammatory disease is selected from the group consisting of inflammation associated with an infection (such as septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-associated hyperacute rejection, nephritis, cytokine- or chemokine-induced lung injury, non-allergic asthma, allergic asthma, allergic rhinitis, atopic dermatitis, cystic fibrosis, pulmonary fibrosis, inflammatory bowel disease, Crohn's disease, and ulcerative colitis.

14. 13. The isolated or recombinant peptide molecule of claim 11 or 12 for use according to claim 11 or 12, wherein the inflammatory disease is acute organ failure associated with surgery, such as acute kidney injury, acute respiratory distress syndrome, associated with surgical interventions, such as cardiac surgery, coronary artery bypass, aortic aneurysm, valve surgery, kidney disease, coronary artery bypass graft surgery, thoracic surgery including but not limited to aortic root and ascending aortic aneurysm repair surgery.

15. 11. The isolated or recombinant peptide molecule of claim 10 for use in the treatment and / or prevention of chronic obstructive pulmonary disease.

16. 11. The isolated or recombinant peptide molecule of claim 10 for use in the treatment and / or prevention of an autoimmune disease.

17. 17. The isolated or recombinant peptide molecule of claim 16 for use in accordance with claim 16, wherein the autoimmune disease is selected from the group consisting of hemolytic anemia, thrombocytopenia, pernicious anemia, Addison's disease, autoimmune diabetes, insulin-dependent diabetes mellitus, myasthenia gravis, rheumatoid arthritis, systemic lupus erythematosus, atherosclerosis, autoimmune encephalitis, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, myasthenia gravis, graft-versus-host disease, and autoimmune inflammatory eye disease.

18. 11. The isolated or recombinant peptide molecule of claim 10 for use in the treatment and / or prevention of an allergic disease.

19. Allergic diseases include eczema, dermatitis, allergic rhinitis (hay fever), allergic airway disease, hypereosinophilic syndrome, contact dermatitis; respiratory diseases such as asthma, including allergic asthma and intrinsic asthma, characterized by eosinophilic airway inflammation and airway hyperresponsiveness, allergic bronchopulmonary aspergillosis, eosinophilic pneumonia, allergic bronchitis bronchiectasis, occupational asthma, airway hyperresponsiveness syndrome, interstitial lung disease, hypereosinophilic syndrome, pulmonary parasitosis; anaphylaxis.

19. The isolated or recombinant peptide molecule of claim 18 for use in accordance with claim 18, wherein the peptide molecule is selected from the group consisting of rash, rash, rashes ...

20. A pharmaceutical composition comprising a peptide molecule according to any one of claims 1 to 9 and one or more pharma- ceutically acceptable excipients.

21. 20. Use of an isolated or recombinant peptide molecule according to any one of claims 1 to 19 in the manufacture of a medicament for the prevention and / or treatment of a disease according to any one of claims 11 to 19 in a patient.

22. 22. The use of claim 21, wherein the isolated or recombinant peptide molecule is in combination with one or more therapeutic agents.

23. 23. The use of claim 22, wherein the therapeutic agent is selected from disease modifying drugs, analgesics, bronchodilators, anti-inflammatory drugs, anti-allergy drugs, allergen immunotherapy drugs, antiviral drugs, antibiotics, antibodies, and steroids.

24. 23. The use of claim 22, wherein the therapeutic agent is a bronchodilator.

25. 23. The use of claim 22, wherein the therapeutic agent is selected from corticosteroids, anti-leukotrienes, cytokine monoclonal antibodies, and theophylline.

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