Treatment methods for relapsing-remitting diseases

Abstract

To provide a pharmaceutical composition for providing and maintaining therapeutic remission that persists beyond their pharmacokinetic coverage for relapsing-remitting disease.SOLUTION: A pharmaceutical composition of the present invention comprises, for example, one or more doses of an effective amount of a peptide molecule comprising a variant of, or consisting of, the amino acid sequence of DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD comprising one or two amino acid substitutions.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to methods for the acute short-term treatment of relapsing-remitting diseases, and more particularly to methods using peptides derived from the polypeptide chaperonin 60.1 from Mycobacterium tuberculosis, as well as peptide molecules and pharmaceutical compositions for use in such methods.

[0002] Heat shock polypeptides are a family of molecules present in all living organisms, and their function is to assist in the biological processing and stability of biomolecules (Zugel & Kauffman (1999) Role of heat shock polypeptides in protection from and pathogenesis of infectious diseases. Clin. Microbiol. Rev. (12)1: 19-39; Ranford et al. (2000) Chaperonins are cell signalling polypeptides: - the unfolding biology of molecular chaperones. Exp. Rev. Mol. Med., 15 September, www.ermn.cbcu.cam.ac.uk / ).

[0003] Mycobacterium tuberculosis produces a polypeptide, chaperonin 60.1 (Cpn60.1), which is named based on its amino acid sequence identity with other known chaperonins. Other Mycobacterium tuberculosis chaperonin polypeptides include chaperonin 10 (Cpn10) and chaperonin 60.2 (Cpn60.2). Cpn60.2 shows 59.6% amino acid sequence identity to Cpn60.1, and Cpn10 shows 65.6% nucleic acid sequence identity.

[0004] International patent application, publication number WO02 / 040037, discloses a pharmaceutical composition containing Cpn60.1 (MtCpn60.1) derived from Mycobacterium tuberculosis, as well as a nucleic acid molecule encoding MtCpn60.1. The application also discloses several specific peptide fragments that can be derived from full-length polypeptides. Various therapeutic uses of these molecules are also disclosed, including the treatment and / or prevention of autoimmune diseases, allergic diseases, diseases represented by Th2-type immune responses, and eosinophilia-related diseases.

[0005] International patent application, publication number WO2009 / 106819, discloses a series of novel peptides derived from MtCpn60.1, including a peptide (named "peptide 4") having the amino acid sequence: DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 1). Peptide 4 exhibits anti-inflammatory activity and has been shown to significantly reduce eosinophil recruitment in an animal model of allergic airway inflammation.

[0006] Another patent application disclosed a specific small fragment of Sequence ID No. 1 that exhibits enhanced biological activity, particularly its ability to suppress extravasation of leukocytes. This peptide is particularly suitable for pharmaceutical development because it has a relatively short amino acid chain length, which is advantageous for preparation and isolation in high yield. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 02 / 040037 Pamphlet [Patent Document 2] International Publication No. 2009 / 106819 Pamphlet [Non-patent literature]

[0008] [Non-Patent Document 1] Zugel & Kauffman (1999) Role of heat shock polypeptides in protection from and pathogenesis of infectious diseases. Clin. Microbiol. Rev. (12)1: 19-39; Ranford et al. [Non-Patent Document 2] (2000) Chaperonins are cell signaling polypeptides: - the unfolding biology of molecular chaperones. Exp. Rev. Mol. Med., 15 September [Overview of the project]

[0009] However, prior art only discloses the use of such peptides in the chronic, long-term treatment of inflammatory diseases. Such treatments require continuous administration of the peptide, as well as the maintenance of detectable peptide concentrations in plasma, in order to achieve therapeutic effects. This means that patients need to receive peptides for extended periods without interruption, increasing the likelihood of medication compliance problems and increasing the drug burden on patients. Therefore, there is a need for acute, short-term treatments that can be administered at the onset or relapse of the disease to induce remission, but do not require further continuous administration to maintain remission. This invention is based on the unexpected finding that chaperonin peptides may be administered intermittently to obtain therapeutic effects, such as anti-inflammatory effects, in contrast to previously accepted conventions. The observed effect is considered "disease-modifying" insofar as symptoms are minimized after administration of the desired peptide alone.

[0010] Thus, in a first embodiment, the present invention provides a method for acute short-term treatment of relapsing-remitting disease, the method comprising administering to a subject in need of treatment, in response to or at the time of relapse, an effective amount of a peptide molecule comprising, or consisting of, an amino acid sequence selected from one of groups (i) to (xv), the amino acid sequence thereof, in one or more doses, the method causing remission of the disease: (i) DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD (Sequence ID 1); (ii) XHGLNVNTLSYGD (Sequence ID 2) sequence, wherein X is absent in the sequence, or selected from the group consisting of a β-alanine residue, 8-amino-3,6-dioxaoctanoic acid, and an acetyl group; or its variant comprising one or more of ii(i) to ii(iii); ii(i) One or more amino acid residues are of type D, ii(ii) GLNVNTLSYGD is upside down, or ii(iii) The carboxy-terminal amino acid residue has been converted to a primary carboxamide group; (iii) DGSVVVNKVSEL-NH2 (Sequence ID 3); (iv) SELPAGHGLNVNTLSYGDLAAD (Sequence ID 4): (v) SELPAGHGLNVNTLS (Sequence ID 5); (vi) PAGHGLNVNTLS-NH2 (Sequence ID 6); (vii) VVVNKVSELPAGHGLNVNTLSYGDLAAD (Sequence ID 7); (viii) NKVSELPAGHGLNVNTLSYGDLAAD (Sequence ID 8); (ix) PAGHGLNVNTLSYGDLAAD (Sequence ID 9); (x) HGLNVNTLSYGDLAAD (Sequence ID 10); (xi) DGSVVVNKVSELPAGH (Sequence ID 11); (xii) GLNVNTLSYGDLAAD (Sequence ID 12); (xiii) DGSVVVNKVS (Sequence ID 13); (xiv) NTLSYGDLAAD (Sequence No. 14); and (xv) A polypeptide sequence that has more than 85%, 90%, or 95% identity with any of (i) through (xiv) and has equivalent function to any of (i) through (xiv).

[0011] This invention relates to a method for the acute short-term treatment of relapsing-remitting diseases. The inventors have surprisingly found that, once an initial dose is administered, a long-term therapeutic effect may be achieved without the need for continuous or long-term regular administration of the drug. The method of this invention causes disease modification. The agents of this invention are considered to modify (correct toward improvement) the underlying disease or condition, rather than merely treating the symptoms of the disease or condition. The peptides of this invention are disease modifiers, that is, agents that provide sustained therapeutic utility beyond the pharmacokinetic range. The term "disease modifier" was originally used in relation to drugs for treating rheumatoid arthritis, disease-modifying antirheumatic drugs, or so-called "DMARDs." However, "disease modification" is now more commonly used in medicine and pharmacy and is not limited to rheumatoid arthritis. Rather, those skilled in the art will know that disease modification is a term used in relation to the treatment of a variety of diseases. For example, disease modification is also used in relation to the treatment of asthma. For example, Lancet Respiratory Medicine, “Clinical trial research in focus: do trials prepare us to deliver precision medicine in those with severe asthma?”, Brightling (2017), Vol 5 February 2017, 92-95, listed “New treatments for asthma need to be more ambitious to achieve complete disease remission, disease modification, and cure.” as a key finding.In addition, The Lancet Commission: “After asthma: redefining airways diseases”, Ian D Pavord et al, September 11, 2017, S0140-6736(17)30879-6 lists one of its seven recommendations as “Move beyond a disease control-based approach for asthma treatment · Direct resources toward primary prevention strategies (asthma prevention) and disease modifying interventions (asthma cure).”

[0012] As used herein, the term “treatment” means reducing, alleviating, or eliminating one or more symptoms of a disease being treated compared to the symptoms before treatment. The term “acute short-term treatment” is used to mean that the peptide is administered at the time of a relapse or during a relapse period of the disease, but does not need to be administered continuously. In particular, it is considered that the peptide does not need to be administered during periods of remission of the disease. Therefore, the “acute short-term treatment” of the present invention can be distinguished from known treatments for relapsing-remitting diseases that result in chronic long-term treatments, where the drug needs to be administered continuously and without interruption during treatment. Providing acute short-term treatment offers significant benefits to patients. Because the peptide of the present invention only needs to be administered over a short period, side effects such as injection site reactions are reduced. In addition, during periods of remission, patients can enjoy an improved lifestyle without having to remember their medication schedule.

[0013] While not bound by theory, the peptides of the present invention are understood to not only affect the symptoms of a pathological condition or disease, but rather to modify the underlying pathological condition or disease itself. Therefore, administration of the peptides of the present invention has long-term effects.

[0014] In one embodiment, disease remission is maintained without the need for additional peptide doses. Notably, acute short-term treatment is thought to involve administering an effective amount of peptide molecules once or multiple times at the time of disease relapse or during a relapse period. In some cases, one or more doses of peptide may be required during the remission period. However, continued administration of an effective amount of peptide is not necessary. Preferably, no additional doses are required during remission.

[0015] In a particularly preferred embodiment, a single dose of the peptide molecule is administered to the patient.

[0016] In another embodiment, two or more doses, preferably three, are administered over a short period, for example, over a period of 1, 3, 28, 56, or 112 days. The time interval between doses to the subject can be 3 hours, 1 day, 14 days, 28 days, or 56 days after the previous dose.

[0017] Remission typically includes a decrease, reduction or disappearance of one or more symptoms of a disease. Typically, remission or clinical remission includes a period without symptoms associated with a relapsing-remitting disease, or a period during which the severity and / or number of symptoms associated with that disease have decreased. Symptoms associated with a condition, disease or disorder include any clinical symptoms or laboratory findings associated with the disease or disorder. Thus, clinical remission can be evaluated by clinicians and researchers according to appropriate measures or remission criteria well known in the medical field, which vary depending on the disease. In contrast, disease relapse can be defined as an increase or appearance of one or more symptoms of the disease. Of course, the symptoms that decrease, reduce or disappear will depend on the individual relapsing-remitting disease being treated. For example, symptoms of asthma include shortness of breath; difficulty breathing; chest tightness; coughing; reduced lung capacity; sleep disturbances due to shortness of breath, coughing or wheezing; a whistling-like wheezing sound when inhaling; and episodes of coughing or wheezing that are worsened by respiratory viruses such as colds or influenza. Furthermore, symptoms can include hospitalization, loss of employment / school attendance, or death.

[0018] The reduction or disappearance of one or more symptoms is typically a significant reduction or disappearance of one or more symptoms confirmed by a physician. The symptoms of relapsing-remitting diseases can be evaluated and quantified by well-known diagnostic tests. For example, lung function tests such as spirometry, and the methacholine challenge test can be used to quantify asthma symptoms by the ACQ score. The ACQ is a simple questionnaire for evaluating the effectiveness of asthma management, as well as the natural changes in asthma management, or changes resulting from treatment. The ACQ has a multidimensional construct that evaluates symptoms (5-item self-administered), use of bronchodilators for rescue (1-item self-administered), and the forced expiratory volume in one second (FEV1) (1-item) entered by clinic staff (Juniper EF, O'Byrne PM, Guyatt GH, Ferrie PJ, King DR. Development and validation of a questionnaire to measure asthma control. Eur Respir J 1999; 14: 902-907).

[0019] It is also possible to define not only a clinical definition of remission, but also a biological or mechanistic definition of remission. In particularly preferred embodiments, the disease is accompanied by an increase in eosinophils and / or an increase in neutrophils. In this case, remission includes a significant decrease in the number of neutrophils and / or eosinophils transported to the site of inflammation in a human or animal subject compared to a control subject not administered the peptide molecule. When the disease is a lung disease, remission includes a significant decrease in the number of neutrophils and / or eosinophils mobilized in the lung or found in the circulatory system.

[0020] Remission may be accompanied by a significant decrease in lymphocyte count or a significant increase in macrophage count in human subjects compared to control subjects. Remission may also be accompanied by a significant change in the levels of one or more inflammatory markers, such as cytokines like IL-4, IL-5, IL-10, or IL-13, in human subjects compared to control subjects. Remission may include a significant increase in the amount of IL-10 in human subjects compared to control subjects. Remission may include a significant decrease in the amounts of IL-4, IL-5, or IL-13 in human subjects compared to control subjects.

[0021] Relapsing-remitting disease is any disease that has one or more periods of relapse, with each relapse followed by a period of remission.

[0022] During periods without these symptoms, i.e., remission, patients do not require therapeutic peptides at quantifiable blood concentrations. In a preferred embodiment, remission is maintained when the plasma peptide concentration is below the lower limit of quantification. This lower limit of quantification may vary depending on the detection method employed. Typically, plasma peptide concentrations are undetectable at blood concentrations (circulating levels) below 40 ng / mL, e.g., below 30 ng / mL or 20 ng / mL. A typical method for measuring plasma peptide concentrations is high-resolution accurate mass (HRAM) LC-MS / MS.

[0023] In a preferred embodiment, the period of disease remission is at least 7 days, for example, 14 days, at least 28 days, and more preferably at least 6 months, after the concentration of peptide molecules in the subject's plasma becomes undetectable.

[0024] In another embodiment, the period of disease remission is at least 7 days after the final dose of the peptide, but may be at least 14 days, at least 28 days, or at least 6 months.

[0025] Typically, relapsing-remitting diseases are inflammatory diseases. Preferably, the disease is selected from a group consisting of allergic inflammatory diseases such as asthma, Crohn's disease, atopic dermatitis and rhinitis, rheumatoid arthritis, and inflammatory bowel disease.

[0026] A key advantage of the peptide molecules of the present invention is that they are effective therapeutic agents for neutrophil-related diseases such as severe asthma, cystic fibrosis, bronchiectasis (including non-CF), pulmonary hypertension, pulmonary fibrosis, and acute respiratory distress syndrome, inflammatory bowel diseases including ulcerative colitis and Crohn's disease, and chronic obstructive pulmonary disease (COPD). Furthermore, neutrophil-related diseases can include asthma, gout attacks, glomerulonephritis, rheumatic fever, collagen vascular diseases and hypersensitivity reactions and metabolic diseases, such as diabetic ketoacidosis, pre-eclampsia, and uremia, particularly with uremic pericarditis, neutrophil-dominant collagen diseases, Gaucher disease, Cushing's syndrome, myelofibrosis, neoplastic neutrophilia, polycythemia vera, psoriasis, and inflammatory bowel disease. Other examples include Wegner vasculitis, cystic fibrosis, Sjögren's syndrome, chronic rejection of transplants, type 1 diabetes, graft-versus-host disease, thyroiditis, spondyloarthritis, ankylosing spondylitis, uveitis, and polychondritis or scleroderma.

[0027] In other embodiments, the present invention provides the use of the peptides described herein when the relapsing-remitting disease is an autoimmune disease. Examples of autoimmune diseases that can be prevented and / or treated by the peptide molecules of the present invention include hemolytic anemia, thrombocytopenia, pernicious anemia, Addison's disease, autoimmune diabetes, insulin-dependent diabetes, myasthenia gravis, rheumatoid arthritis, systemic lupus erythematosus, atherosclerosis, autoimmune encephalitis, connective tissue disease, multiple sclerosis (including relapsing multiple sclerosis), autoimmune pneumonia, Guillain-Barré syndrome, autoimmune thyroiditis, graft-versus-host disease, and autoimmune inflammatory eye disease. Preferred autoimmune diseases include rheumatoid arthritis and systemic lupus erythematosus.

[0028] In other embodiments, the present invention provides the use of the peptides described herein for the remission of allergic diseases. Examples of allergic diseases that may be improved or alleviated by treatment with the peptide molecules of the present invention include eczema, atopic dermatitis, allergic rhinitis (hay fever), allergic airway diseases, respiratory diseases such as asthma including allergic asthma and endogenous asthma characterized by eosinophilic syndrome, eosinophilic airway inflammation and airway hyperresponsiveness, allergic bronchopulmonary aspergillosis, eosinophilic pneumonia, allergic bronchitis bronchiectasis, interstitial lung disease, eosinophilic syndrome, urticaria, angioedema, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, and giant papillary conjunctivitis. Preferred allergic diseases include asthma, allergic rhinitis, and atopic dermatitis. In another embodiment, the above diseases include viral exacerbations of allergic diseases such as asthma. In another embodiment, the above-mentioned diseases also include exacerbations associated with bacterial infections.

[0029] Preferably, remission includes a reduction, alleviation, or disappearance of one or more symptoms of the disease over a period significantly exceeding the pharmacokinetic half-life of the peptide. This typically means a significant decrease in the severity of the disease over a period significantly exceeding the pharmacokinetic half-life of the peptide therapeutic agent.

[0030] The peptide can be used in accordance with the present invention when the patient is receiving one or more other therapeutic agents, or when the peptide is administered in combination with one or more therapeutic agents. The therapeutic agents can be selected from, but are not limited to, disease-modulating agents such as immunomodulators, analgesics, bronchodilators, anti-inflammatory agents, anti-allergic agents, allergen immunotherapies, antiviral agents, antibiotics, antibodies, steroids, and drugs commonly used to treat relapsing-remitting diseases according to the present invention.

[0031] Disease-modifying agents include, for example, hydroxychloroquine, sulfasalazine, leflunomide, methotrexate, and minocycline, as well as TNFα-targeting biologics such as abatacept, adalimumab, etanercept, infliximab, and golimumab, or immunomodulators such as alemtuzumab, interferon β-1b, β-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 opioids.

[0032] Anti-inflammatory drugs include leukotriene receptor antagonists, theophylline, selective phosphodiesterase (PDE) 4 inhibitors such as roflumilast, dual PDE3 / 4 inhibitors such as RPL 554, and low-dose, medium-dose, 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) and antibodies that modify cytokine signaling pathways (e.g., the 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 is preferred if one or more additional therapeutic agents are selected from corticosteroids, anti-leukotrienes (antileukotriene agents), cytokine monoclonal antibodies, or theophylline. The use is also preferred if the additional agent is a bronchodilator. Preferred bronchodilators include short-acting β2 agonists, such as salbutamol; long-acting β2 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 acridinium bromide, tiotropium bromide, and glycopyrronium bromide.

[0033] The peptides of the present invention are synthesized chemically or by recombinant means and possess several chemical, structural, and functional properties distinct from full-length chaperonin 60.1.

[0034] A "functionally equivalent" peptide means any peptide and / or variant or fragment thereof that has a function (e.g., biological activity) identical or substantially similar to any function exhibited by one or more of the defined amino acid sequences (i) through (xiv), or any function resulting from those sequences. For example, a peptide consisting of the amino acid sequence defined in (i) reduces the transport of immune cells such as eosinophils and / or neutrophils to the site of inflammation, and therefore can be used to prevent and / or treat a variety of diseases and disorders, such as asthma, rheumatoid arthritis, and inflammatory bowel disease (including ulcerative colitis and Crohn's disease). Functional equivalence with respect to specific biological activity can be evaluated using conventional models and methods; for example, by measuring the influx of inflammogen-induced immune cells, such as eosinophils or neutrophils, into the lungs in sensitized (inflammogen-ovalbumin / house dust mite) or naive (inflammogen-LPS) animals.

[0035] The peptides shall have an amino acid sequence that is 80% or more, 90% or more, or 95% or more identical to sequences (i) to (xiv) above. These peptide molecules may be produced, for example, using recombinant DNA technology, and may differ by amino acid insertions, deletions, and substitutions. Guidelines for determining which amino acid residues can be substituted, added, or deleted without losing the desired activity can be found by comparing the sequence of the individual polypeptide with the sequence of the homologous peptide and minimizing the number of amino acid sequence changes occurring in highly homologous regions (conserved regions), or by replacing amino acids with the consensus sequence.

[0036] Alternatively, recombinant variants encoding these identical or similar polypeptides can be synthesized or selected by utilizing the "redundancy (degeneracy)" of the genetic code. Various codon substitutions, such as silent changes resulting in different restriction enzyme sites, can be introduced to optimize cloning into plasmids or viral vectors, or expression in specific prokaryotic or eukaryotic cell lines. While polynucleotide mutations are reflected in the polypeptide, domains from other peptides can also be added to the polypeptide to modify the properties of any portion of the polypeptide, altering characteristics such as ligand-binding affinity, interchain affinity, or degradation / turnover rate.

[0037] 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 based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues involved. For example, nonpolar (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" are preferably in the range of about 1 to 10 amino acids, but more preferably 1 to 5 amino acids, for example, 1, 2, 3, 4, or 5 amino acids. Acceptable mutations can be experimentally confirmed by systematically inserting, deleting, or substituting amino acids into polypeptide molecules using recombinant DNA technology or synthetic methods such as solid-phase synthesis, and then assaying the biological activity of the resulting recombinant variants.

[0038] Alternatively, if functional modification is desired, modified polypeptides can be created by designing insertions, deletions, or non-conservative mutations. Such mutations may alter, for example, one or more biological functions or biochemical properties of the polypeptides of the present invention. For example, such mutations may alter polypeptide properties such as ligand-binding affinity, interchain affinity, or degradation / turnover rate. Furthermore, such mutations may be selected to produce polypeptides better suited for synthetic production, expression in selected host cells for expression, scale-up, etc.

[0039] The present invention also includes peptide fragments capable of exhibiting biological activity. 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, depending on the application, including increasing the titer of the protein binding site.

[0040] "Identity" means the number or percentage (depending on the display of the results) of amino acid residues or nucleic acid residues in a candidate sequence that are identical to the amino acid residues or nucleic acid residues in the given sequence, after aligning the sequences and introducing gaps where necessary to achieve the maximum percentage sequence identity. Conservative substitutions are not considered part of sequence identity.

[0041] The percentage sequence identity between two polynucleotides or polypeptides can be determined using a suitable computer program, such as the GAP program from the University of Wisconsin Genetic Computing Group. Naturally, the percentage identity is calculated for optimally aligned polypeptide sequences. Alignment can also be performed using the Clustal W program (Thompson et al., (1994) Nucleic Acids Res. 22, 4673-80). The parameters used are 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.

[0042] Sequence identity can be determined, for example, using the Jotun-Hein method (Hein, J. (1990) Methods Enzymol. 183:626-645). Sequence identity can also be determined by other methods known in the art, for example, by varying the hybridization conditions.

[0043] The peptides of the present invention can be prepared and / or isolated using conventional methods known in the art. For example, by conventional methods or by using an automated solid-phase synthesizer, such as those described in I. Coin, Nature Protocols, 2007, 2, 3247-3256, for example, by solution synthesis or solid-phase synthesis. 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.

[0044] In a preferred embodiment, the peptide molecule consists of an amino acid sequence selected from one of groups (a) to (s): (a) DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD (Sequence ID 1); (b) XHGLNVNTLSYGD-NH2 (Sequence ID 15); (c) XdGysltnvnlGh-NH2 (Sequence ID 16); (d) XhGlnvntlsyGd-NH2 (Sequence ID 17); (e) hGLNVNTLSYGd-NH2 (Sequence ID 18); (f) HGLNVNTLSYGd-NH2 (Sequence ID 19); (g) hGLNVNTLSYGD-NH2 (Sequence ID 20); (h) DGSVVVNKVSEL-NH2 (Sequence ID 3); (i) SELPAGHGLNVNTLSYGDLAAD (Sequence ID 4); (j) SELPAGHGLNVNTLS (Sequence ID 5); (k) PAGHGLNVNTLS-NH2 (Sequence ID 6); (l) VVVNKVSELPAGHGLNVNTLSYGDLAAD (Sequence ID 7); (m) NKVSELPAGHGLNVNTLSYGDLAAD (Sequence ID 8); (n) PAGHGLNVNTLSYGDLAAD (Sequence ID 9); (o) HGLNVNTLSYGDLAAD (Sequence ID 10); (p) DGSVVVNKVSELPAGH (Sequence ID 11); (q) GLNVNTLSYGDLAAD (Sequence ID 12); (r) DGSVVVNKVS (sequence number 13); and (s) NTLSYGDLAAD (Sequence ID 14); In the formula, uppercase letters indicate L-amino acid residues, lowercase letters indicate D-amino acid residues, and X is either absent or selected from the group consisting of β-alanine residues, 8-amino-3,6-dioxaoctanoic acid, and acetyl groups.

[0045] The nomenclature of amino acids and peptide derivatives follows IUPAC-IUB rules (J. Peptide Sci. 1999, 5, 465-471). D-amino acids are represented by lowercase abbreviations; for example, L-alanine is represented as Ala or A, and D-alanine as ala or a.

[0046] The peptide of the present invention preferably consists of 5 to 50 or 5 to 40 amino acid residues, and more preferably consists of 5 to 35 or 5 to 20 amino acid residues.

[0047] In a particularly preferred embodiment, the peptide comprises the amino acid sequence DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 1). The peptide is preferably less than 50 amino acid residues long, and most preferably less than 40 amino acid residues long. In a particularly preferred embodiment, the peptide consists of SEQ ID NO: 1, which has 31 amino acid residues.

[0048] In a preferred embodiment, the isolated peptide molecule or recombinant peptide molecule consists of the amino acid sequence HGLNVNTLSYGD-NH2 (SEQ ID NO: 21), or a functionally equivalent fragment or variant thereof.

[0049] In a preferred embodiment, the isolated peptide molecule or recombinant peptide molecule consists of the amino acid sequence bAla-HGLNVNTLSYGD-NH2 (SEQ ID NO: 22), or a functionally equivalent fragment or variant thereof.

[0050] In another preferred embodiment, the isolated peptide molecule or recombinant peptide molecule consists of the amino acid sequence Ac-dGysltnvnlGh-NH2 (SEQ ID NO: 23), Ac-hGlnvntlsyGd-NH2 (SEQ ID NO: 24), or a functionally equivalent fragment or variant thereof.

[0051] In another preferred embodiment, the present invention provides an isolated peptide molecule or a recombinant peptide molecule comprising the amino acid sequence hGLNVNTLSYGd-NH2 (SEQ ID NO: 18), or a functionally equivalent fragment or variant thereof.

[0052] In yet another preferred embodiment, the present invention provides an isolated peptide molecule or a recombinant peptide molecule comprising the amino acid sequence HGLNVNTLSYGd-NH2 (SEQ ID NO: 19) or a functionally equivalent fragment or variant thereof.

[0053] In yet another preferred embodiment, an isolated peptide molecule or recombinant peptide molecule is given, comprising the amino acid sequence hGLNVNTLSYGD-NH2 (SEQ ID NO: 20); or a functionally equivalent fragment or variant thereof.

[0054] In another preferred embodiment, an isolated peptide molecule or a recombinant peptide molecule is given, comprising the amino acid sequence DGSVVVNKVSEL-NH2 (SEQ ID NO: 3); or a functionally equivalent fragment or variant thereof.

[0055] In another embodiment, the present invention provides the peptide molecule described above for use in the method described above.

[0056] Relapsing-remitting diseases can include autoimmune diseases such as rheumatoid arthritis, or inflammatory bowel disease (IBD).

[0057] The peptide molecules and pharmaceutical compositions of the present invention can be introduced into cells by "Trojan peptides." These are a type of polypeptide called penetratin, which has mobility properties and the ability to transport hydrophilic compounds across the cell membrane. This system allows for the direct targeting of oligopeptides to the cytoplasm and nucleus, and can be cell-type nonspecific and highly efficient. See Derossi et al. (1998), Trends Cell Biol 8, 84-87.

[0058] Compounds for use in this invention are understood to include salts. Metabolites and prodrugs are also included. Compounds for use in accordance with this invention also include any isotopic derivatives.

[0059] The compounds described herein can be formulated for administration in an easy-to-use manner. The present invention provides a pharmaceutical composition for use in the above method, comprising the above peptide molecules and one or more pharmaceutically acceptable additives.

[0060] The peptides of the present invention may be used for use in humans or non-human animals, typically mammals.

[0061] Any suitable route of administration can be used. For example, oral, topical, parenteral, intraocular, rectal, vaginal, inhalation, buccal, sublingual, and nasal routes of administration are all considered suitable.

[0062] Pharmaceutical compositions for parenteral administration may be preferred. The peptide molecules and pharmaceutical compositions of the present invention can be administered parenterally, for example, intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intracisionally, intracranially, intramuscularly, or subcutaneously, but may also be administered by drip infusion. They are best used in the form of sterile aqueous solutions, which may contain other substances, such as salts or glucose sufficient to make the solution isotonic with blood. The aqueous solutions should be appropriately buffered as needed (preferably to a pH of 3-9). Preparation of suitable parenteral formulations under sterile conditions can be achieved by standard compounding techniques well known to those skilled in the art. Subcutaneous administration is considered preferred.

[0063] Drugs and pharmaceutical compositions suitable for parenteral administration include sterile injectable aqueous and non-aqueous solutions containing antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the blood of a given recipient; as well as sterile aqueous and non-aqueous suspensions containing suspending agents and thickeners. Drugs and compositions can be supplied in single-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored lyophilized, requiring only the addition of a sterile liquid carrier such as distilled water for injection immediately before use. Injectable solutions and suspensions can be prepared on-site immediately from sterile powders, granules, and tablets as described above.

[0064] The molecules, drugs, and pharmaceutical compositions of the present invention can also be administered intranasally or by inhalation, for example, in the form of a dry powder inhaler or in the form of an aerosol spray from a pressurized container, pump, spray or nebulizer, conveniently administered using carbon dioxide or other suitable gas as a suitable spraying agent, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, hydrofluoroalkanes, such as 1,1,1,2-tetrafluoroethane (HFA 134A3) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3). In the case of a pressurized aerosol, the dose unit can be measured by equipping a valve, and the measured amount can be administered. The pressurized container, pump, spray or nebulizer contains a solution or suspension of the active drug, for example, using a mixture of ethanol and the spraying agent as a solvent, and may also contain a lubricant such as sorbitan trioleate. Capsules and cartridges for inhalers (for example, made of gelatin) can be formulated to contain a mixed powder of the drug of the present invention and a suitable powder base such as lactose or starch.

[0065] The aerosol or dry powder formulation is preferably prepared such that each measured dose or "puff" contains at least 100 pg or 200 pg of the molecule of the present invention for administration to a patient. Naturally, the total daily dose of the aerosol will vary from patient to patient and may be administered as a single dose, but more commonly, it is administered in divided doses throughout the day.

[0066] The peptide molecules and pharmaceutical compositions of the present invention can also be administered orally. The process utilizes the body's natural processes for oral intake of vitamin B12 and / or vitamin D, allowing for the simultaneous delivery of proteins and peptides. 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 the vitamin B12 analog and / or vitamin D analog and the drug, the complex retaining both significant affinity for intrinsic factor (IF) in the vitamin B12 / vitamin D portion of the complex, as well as significant biological activity of the active substance of the complex.

[0067] The peptide molecules and pharmaceutical compositions of the present invention are typically administered in the form of pharmaceutical compositions containing the active ingredient, usually via any parenteral route or intranasally, but may also be administered orally depending on the embodiment. Depending on the disease being treated and the patient, as well as the route of administration, the compositions are administered in various doses.

[0068] In human treatment, the peptide molecules and pharmaceutical compositions of the present invention may be administered alone, but in most cases they are administered in mixture with appropriate pharmaceutical additives, diluents, or bases, which are selected in accordance with the planned route of administration and standard pharmaceutical practice.

[0069] Preferably, the pharmaceutical composition of the present invention is a unit formulation containing a daily dose or unit amount of the active ingredient, a divided daily dose, or an appropriate small portion thereof.

[0070] For example, the peptide molecules and pharmaceutical compositions of the present invention can be administered orally, buccally, or sublingually in the form of tablets, capsules, suppositories, elixirs, solutions, or suspensions (which may contain fragrances or colorants) for immediate release, delayed release, or controlled release. The peptide molecules and pharmaceutical compositions of the present invention can also be administered by intracavernosal injection.

[0071] Such tablets may contain additives 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 granulating binders such as polyvinylpyrrolidone, hydroxypropyl methylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin, and gum arabic. Furthermore, lubricants such as magnesium stearate, stearic acid, and glyceryl behenate may also be included.

[0072] Similar solid compositions may be used as excipients in gelatin capsules. Preferred additives for this purpose include lactose, starch, cellulose, lactose, or high molecular weight polyethylene glycol. For aqueous suspensions and / or elixirs, the drug of the present invention may be mixed with various sweeteners or flavorings, colorants or pigments, emulsifiers and / or suspending agents, and diluents such as water, ethanol, propylene glycol, and glycerin, as well as combinations thereof.

[0073] For oral and parenteral administration to human patients, the daily dose levels of the molecules, drugs, and pharmaceutical compositions of the present invention are typically 200 pg to 100 mg per day for adults, administered as a single dose or in divided doses.

[0074] Therefore, for example, vials, tablets, or capsules of the molecule of the present invention contain 200 pg to 100 mg of the active agent for administration one at a time, two at a time, or three or more at a time, as appropriate. In any case, the physician will determine the most appropriate and precise dosage for the individual patient, which will vary depending on the individual patient's age, weight, and response. The above dosages are examples of average cases. Of course, there may be individual cases where higher or lower dosage ranges are appropriate, and such cases are within the scope of the present invention.

[0075] Alternatively, the molecules, drugs, and pharmaceutical compositions of the present invention may be administered as suppositories or vaginal suppositories, but may also be applied topically as lotions, liquids, creams, gels, ointments, or powders. The molecules, drugs, and pharmaceutical compositions of the present invention may also be administered transdermally, for example, by using skin patches. They may also be administered via intraocular routes, particularly to treat eye diseases.

[0076] For ophthalmic use, the molecules, drugs, and pharmaceutical compositions of the present invention can be formulated as a micronized suspension in pH-adjusted sterile isotonic saline, or preferably as a solution in pH-adjusted sterile isotonic saline, but may be combined with preservatives such as benzalkonium chloride. Alternatively, they may be formulated as an ointment such as petrolatum.

[0077] For topical application to the skin, the molecules, agents, and pharmaceutical compositions of the present invention can be formulated as suitable ointments containing active agents suspended or dissolved in one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene, emulsifying wax, and water. Alternatively, they can be formulated as suitable lotions or creams suspended or dissolved in one or more of the following: mineral oil, sorbitan monostearate, polyethylene glycol, liquid paraffin, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0078] Formulations suitable for topical administration in the oral cavity include lozenges containing the active ingredient in a flavored base, usually sucrose and gum arabic or tragacanth; medicinal drops containing the active ingredient in an inert base such as gelatin and glycerin, or sucrose and gum arabic; and mouthwashes containing the active ingredient in a suitable liquid base.

[0079] For veterinary use, the molecules, drugs, and pharmaceutical compositions of the present invention are administered as formulations that are appropriately accepted according to normal veterinary practice, allowing veterinarians to determine the optimal administration plan and route for individual animals.

[0080] It is advantageous for the preparation to be a pharmaceutical preparation. It is also advantageous for the preparation to be a veterinary preparation.

[0081] Advantageously, according to the present invention, the daily dose level is 10 pg to 100 mg. Preferably, the daily dose level is 20 pg to 50 mg, 20 pg to 10 mg, or 20 pg to 8 mg, and is administered as a single dose or in divided doses.

[0082] Preferred pharmaceutical formulations include those containing an active ingredient in an amount of 0.000001% to 5% by weight. In other words, the ratio of the active ingredient to the other components of the pharmaceutical composition (i.e., the addition of auxiliaries, diluents, and bases) is at least 1:99 by weight (for example, at least 10:90, preferably at least 30:70, and most preferably at least 50:50).

[0083] Preferably, the pharmaceutical composition or agent of the present invention is formulated to allow administration via at least one route, including or selected from the group comprising intranasal, oral, parenteral, topical, ophthalmic, suppository, vaginal suppository, or inhalation routes. Formulations suitable for such routes of administration are well known to those skilled in the art of pharmacy and medicine, and typical formulations are described above and in the accompanying examples.

[0084] A non-limiting example is described below with respect to the following drawings. [Brief explanation of the drawing]

[0085] [Figure 1] Figure 1 shows a schematic diagram of the methodology for house dust mite (HDM) antigen challenge to examine the effect of Sequence ID No. 1. [Figure 2]Figure 2 shows the effect of SEQ ID NO: 1 on lung cell recruitment at day 7 (7 days after the first HDM challenge) in female Balb / c mice sensitized with HDM for 3 weeks. Eosinophil and neutrophil counts were measured in the following groups: saline, saline / HDM, 0.02 μg / kg SEQ ID NO: 1 / HDM, 0.2 μg / kg SEQ ID NO: 1 / HDM, and 2 μg / kg SEQ ID NO: 1 / HDM. [Figure 3] Figure 3 shows the effect of SEQ ID NO: 1 on lung cell recruitment at day 7 post-administration in female Balb / c mice sensitized with HDM treatment for 3 weeks. There is a tendency for increased macrophage recruitment to the lungs associated with the treatment group. [Figure 4] Figure 4 shows the effect of SEQ ID NO: 1 on lung cell recruitment at day 14 (4 hours after the second HDM challenge), corresponding to the disease modification time, in female Balb / c mice sensitized with HDM for 3 weeks. Eosinophil, neutrophil, and lymphocyte counts were measured in the following groups: saline, saline / HDM, 0.02 μg / kg SEQ ID NO: 1 / HDM, 0.2 μg / kg SEQ ID NO: 1 / HDM, and 2 μg / kg SEQ ID NO: 1 / HDM. Details of the statistical analysis are shown in the figure. [Figure 5] Figure 5 shows the effect of SEQ ID NO: 1 on allergy-related cytokine levels in bronchoalveolar lavage fluid analysis at the disease modification time (14 days after administration) in the HDM experiment. The data are for SEQ ID NO: 1 administered intranasally at doses of 0.02 μg / kg, 0.2 μg / kg, and 2 μg / kg. Details of the statistical analysis are shown in the figure. [Figure 6] Figure 6 shows the effect of SEQ ID NO: 1 on the anti-inflammatory cytokine IL-10 levels in bronchoalveolar lavage fluid analysis 7 days after administration. This is related to the increase in macrophage recruitment shown in Figure 3 and is thought to reflect the enhancement of anti-inflammatory immune properties that resolve inflammation by SEQ ID NO: 1. Details of the statistical analysis are shown in the figure. [Figure 7]Figure 7 shows the effect of intravenous administration of SEQ ID NO: 1 on eosinophil infiltration into the lung 10 days after administration, 24 hours after the OVA challenge, and 24 hours after the OVA rechallenge. SEQ ID NO: 1 was administered at a dose of 20 ng / kg prior to each OVA challenge in Balb / c mice sensitized with allergen treatment for one week. [Figure 8] Figure 8 shows the effect of intranasal administration of SEQ ID NO: 3 (5 and 50 ng / mouse) on cell recruitment to the lungs after ovalbumin allergen challenge in ovalbumin-sensitized mice. The total number of recruited cells and the percentage of leukocytes relative to eosinophils are shown. [Figure 9] Figure 9 shows the effect of Sequence ID No. 1 on increasing LPS-stimulated IL-10 secretion in human macrophages. [Figure 10] Figure 10 shows evidence linking the mechanism of action of Sequence ID No. 1 to dendritic cell maturation and T cell maturation / development. [Figure 11-1] Figure 11 shows the effect of Sequence ID No. 1 on reducing the suppression of IL-10 gene expression related to dendritic cell maturation, reaffirming the link between the mechanism of action of Sequence ID No. 1 and the expression / release of the anti-inflammatory cytokine IL-10. A change in expression greater than twofold is considered statistically significant. [Figure 11-2] This is a continuation of Figure 11-1. (Example) [Example 1]

[0086] (Experiment with house dust mites) The purpose of this experiment was to investigate the inhibitory effects of three different concentrations of peptide solutions dissolved in physiological saline on house dust mite (HDM)-induced pneumonia in Balb / C mice using a 3-week intranasal (in) HDM-sensitized model. The results for SEQ ID NO: 1 are shown. This experiment also investigated the time-dependent effects of SEQ ID NO: 1; in addition to 7 days after the HDM challenge, another time point, 4 hours after the second HDM challenge (14 days after the initial HDM challenge), was also examined to evaluate the effect of SEQ ID NO: 1 on the remission of the allergic reaction.

[0087] The HDM challenge model is a well-established and reliable model that induces airway inflammation by intranasal (in) administration of allergens.

[0088] 1. Peptide synthesis The peptide was synthesized and isolated according to the following procedure: Synthesis and purification were performed using an automated fluorenylmethyloxycarbonyl solid-phase peptide synthesis method (Fmoc SPSS). The peptides were synthesized using the Fmoc / t-butyl solid-phase synthesis method on Wang resin derivatized with one of numerous cleavable linkers. Transient N-amino group protection was given by the Fmoc group, with t-butyl ether used to protect the hydroxyl side chains of tyrosine, serine, and threonine, while t-butyl ester protected the side chains of aspartic acid and glutamic acid residues. Histidine and lysine side chains were protected as N-trityl and N-Boc derivatives, respectively, cysteine ​​as an S-trityl derivative, and the guanidine moiety of arginine as a Pbf derivative.

[0089] Immediately after synthesis was complete, the peptide was cleaved from the solid support, and the protecting groups of the side chains were removed by treatment with trifluoroacetic acid (TEA) containing triisobutylsilane and water as scavengers. After removing the TFA and scavengers by evaporation and tritulate in diethyl ether, the peptide was purified by reverse-phase HPLC and then lyophilized. The purified product was then analyzed by reverse-phase HPLC and mass spectrometry.

[0090] 2. Experimental Protocol method A 3-week sensitization challenge model was applied, using n=90 female Balb / C mice that were approximately 6-8 weeks old and weighed about 20-25g at the start of the experiment.

[0091] After 11 days of acclimatization, all mice received in-dose administration of 25 μg (total protein) HDM five days a week for three weeks. HDM sensitization was performed daily, and all intranasal administrations were carried out under mild anesthesia using isoflurane. Two weeks after the last HDM sensitization, all mice received a single in-dose administration of 50 μL of a peptide solution in saline or saline (0.9% w / v sodium chloride) solvent. For SEQ ID NO: 1, the administered solutions were 0.02 μg / kg, 0.2 μg / kg, and 2 μg / kg. Fifteen minutes after administration of the peptide or saline solvent, all mice received a single 50 μL challenge of in-dose saline (0.9% w / v sodium chloride) or 100 μg HDM.

[0092] A portion of the animal group received a second dose of 50 μL in either saline or 100 μg of HDM, four weeks after sensitization. The animals were then euthanized four hours after the second challenge (corresponding to 14 days after the initial administration).

[0093] All mice were euthanized by intraperitoneal (IP) overdose of pentobarbital sodium (200 mg / mL). Death was confirmed by cervical dislocation.

[0094] Post-mortem sample collection Bronchoalveolar lavage solution (BAL solution) The lungs were lavaged through the trachea using 1 mL of BAL (bronchial alveolar lavage) solution. The samples were placed on ice water (4°C), and cell type counting was performed using a Sysmex XT2000i hemocytometer. The BAL samples were then centrifuged at 1300 rcf for 7 minutes. 200 μL of the BAL supernatant sample was placed in a polypropylene U-bottom 96-well plate and subsequently frozen at -20°C for cytokine analysis using a standard MSD protocol.

[0095] Data Analysis Data analysis was performed using Graphpad PRISM 6. Statistical tests applied to determine statistical significance were either post-hoc tests or independent t-tests after analysis of variance (ANOVA).

[0096] 3. Results BAL cell influx - as of day 7 Cellular infiltration analysis showed increased neutrophil and eosinophil levels in all HDM-treated mice compared to the saline / saline group. Animals treated with 2 μg / kg of SEQ ID NO: 1 showed decreased neutrophil and eosinophil levels compared to saline / HDM, although this was not statistically significant (Figure 2).

[0097] The analysis also shows an increasing trend in invasive macrophages associated with the SEQ ID NO: 1 treatment group (Figure 3).

[0098] BAL cell influx - as of day 14 Animals treated with 2 μg / kg of SEQ ID NO: 1 showed a significant decrease in neutrophil, eosinophil, and lymphocyte levels compared to saline / HDM.

[0099] Cellular influx levels of eosinophils, neutrophils, and lymphocytes were significantly higher in animals administered with saline / HDM compared to those administered with saline / saline, across all administration routes.

[0100] Animals administered 2 μg / kg of Sequence ID No. 1 showed significantly lower neutrophil, eosinophil, and lymphocyte levels compared to animals administered with saline / HDM (Figure 4).

[0101] BAL cytokine release At 2 μg / kg, Sequence ID No. 1 showed significant suppression of IL-4, IL-5, and IL-13 levels compared to physiological saline / HDM at day 14 (Figure 5).

[0102] Sequence ID 1 showed a significant dose-responsive release of the anti-inflammatory cytokine IL-10 compared to saline / HDM at day 7 (Figure 6).

[0103] 4. Conclusion The animals responded to the second HDM challenge, and statistically significant differences were observed between positive and negative control animals. Four hours after the second HDM challenge and 14 days after administration, statistically significant inhibition of BAL eosinophil, neutrophil, and lymphocyte influx was observed with the highest dose of SEQ ID NO: 1 (2 μg / kg / HDM) compared to animals receiving saline / HDM.

[0104] An increasing trend in macrophage influx was observed in the SEQ ID NO: 1 treatment group at day 7 compared to animals treated with saline / HDM. At this point, animals treated with SEQ ID NO: 1 showed a dose-dependent increase in the anti-inflammatory cytokine IL-10 in the BAL. IL-10 is released from macrophages. These data, taken together, suggest a transition to an anti-inflammatory phenotype that resolves inflammation, which may explain the data at day 14 to some extent.

[0105] Fourteen days after administration and four hours after the challenge, allergy-related Th2 cytokines, IL-4, IL-5, and IL-13 in BAL were suppressed by 2 μg / kg SEQ ID NO: 1 compared to the saline / HDM group.

[0106] These results demonstrate that a single dose of the peptide (shown here as SEQ ID NO: 1) produces long-term effects, regardless of the blood levels at which the peptide is no longer present. [Example 2]

[0107] Ovalbumin Experiment Figures 7 and 8 show the suppression of allergic inflammation in a mouse ovalbumin model by intravenous administration of SEQ ID NO: 1 and intranasal administration of SEQ ID NO: 3, respectively.

[0108] method: In this experiment, female BALB / c mice were immunized intraperitoneally via the peritoneal route with 30 μg of chicken egg albumin absorbed in saturated aluminum hydroxide solution (2.5 mg / ml). Controls received aluminum hydroxide alone. After one week (day 7), OVA infusions were repeated. On day 15, all animals were administered antigen with a 3% ovalbumin spray solution once daily for 25 minutes for three consecutive days. Ten minutes before each ovalbumin challenge, SEQ ID NO: 1 was diluted in sterile saline and administered intravenously at 20 ng / kg (25 μl / mouse). In another series of experiments, ten minutes before each ovalbumin challenge, SEQ ID NO: 3 was diluted in sterile saline and administered intranasally at concentrations of 5 and 50 pg / mouse. Controls received the solvent alone.

[0109] Ten days after the final OVA challenge, the animals treated with Sequence ID No. 1 underwent another three consecutive days of OVA re-challenges. Treatment with Sequence ID No. 1 was not involved in this stage (Figure 7).

[0110] 24 hours after the challenge, mice were euthanized by an overdose of urethane (25% solution ip), a cannula was inserted into the exposed trachea, and 0.5 ml of physiological saline was injected into the lungs three times. The fluid was then collected as bronchoalveolar lavage (BAL) fluid. A portion (50 μL) of the BAL 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 using a modified Neubauer hemocytometer. To determine the leukocyte percentage, cytospin specimens were prepared from 100 μL of BAL fluid centrifuged at 1000 rpm for 1 minute using a Shandon Cytospin 2 (Shandon Southern Instruments, Sewickley, PA, USA) at room temperature. Cells were stained with Diff Quick (DADE Behring, Germany), and a total of 100 cells were counted to determine the proportions of neutrophils, eosinophils, and monocytes using standard morphological criteria.

[0111] Results and conclusions: The total number of cells counted in BAL fluid collected from OVA-challenged mice 24 hours after the final ovalbumin (OVA) challenge was significantly higher than that of control mice, reflecting a significant increase in the number of eosinophils recruited to the airways of OVA-challenged mice. The intensity of the inflammatory response was significantly reduced in animals treated intravenously with SEQ ID NO: 1 before each OVA challenge (Figure 7).

[0112] Ten days after the final OVA challenge, the animals underwent another three consecutive OVA challenges. Treatment with SEQ ID NO: 1 was not involved in this stage. The results show a decrease in the number of eosinophils in the lungs responsive to OVA, ten days after the final OVA challenge. Treatment with SEQ ID NO: 1 was effective in suppressing eosinophil recruitment at this late stage, despite the fact that the blood levels of the peptide were no longer detectable (Figure 7).

[0113] Figure 8 shows the effect of intranasal administration of SEQ ID NO: 3 in an OVA model 24 hours after the final allergen challenge. The intensity of the inflammatory response was significantly reduced in animals treated with intranasal administration of 50 ng / mouse SEQ ID NO: 3 before each OVA challenge, in terms of both total cell count and eosinophil count. [Example 3]

[0114] Effect of SEQ ID NO: 1 on IL-10 release from LPS-stimulated macrophages Figure 9 shows the enhancement of IL-10 secretion in LPS-stimulated macrophages by Sequence ID No. 1.

[0115] method: Human THP-1 mononuclear cells were differentiated into macrophages (100K cells / well) on a 96-well plate for 48 hours using 50 nM phorbol myristart acetate.

[0116] Macrophages increase the concentration of bacterial lipopolysaccharide (LPS 10) in the presence or absence of SEQ ID NO: 1 (1nM). -6 Cells were stimulated with LPS (~10 μg / ml). 30 μl of cell medium was sampled 6 hours after LPS stimulation, and IL-10 alphaLISA was performed to measure IL-10 release.

[0117] Results and conclusions: LPS stimulated IL-10 release from THP-1 differentiated macrophages. Treatment with SEQ ID NO: 1 (1 nM) enhanced IL-10 release at the highest LPS dose (1–10 μg / ml; Figure 9). [Example 4]

[0118] Affymetrix microarray analysis based on lung tissue samples Figure 10 shows the results of Affymetrix microarray analysis of lung tissue obtained from the HDM experiment in Example 1, suggesting that Sequence ID No. 1 is related to dendritic cell maturation and T cell activation / development.

[0119] method Twenty-five mouse tissue FFPF (formalin-fixed paraffin-embedded) samples from the experiment in Example 1 were analyzed after being exposed to one of four treatments (physiological saline, SEQ ID NO: 1 0.02 μg / kg, SEQ ID NO: 1 2 μg / kg, or positive control fluticasone furoate) at 4 hours or 7 days after stimulation with house dust mites (HDM, 100 μg). One physiological saline sample was included at each time point as a solvent control without HDM stimulation.

[0120] RNA was extracted from these samples, and the properties of each sample were evaluated, confirming, as expected, that they matched other FFPE samples. Sufficient RNA was obtained from the samples to proceed with cDNA synthesis. 29 cDNAs were synthesized using the Nugen Ovation® FFPE WTA System.

[0121] The cDNA passed typical QC in FFPE, proceeding to labeling and hybridization with the GeneChip® Mouse Genome 430 2.0 Array.

[0122] Results and conclusions The results were analyzed in a blinded manner based on two independent bioinformatics groups, and the conclusions were similar: namely, the mechanism of action of Sequence ID No. 1 is related to dendritic cell maturation, as well as T cell activation and development (Figure 10). [Example 5]

[0123] The effect of Sequence ID No. 1 on gene expression related to dendritic cell maturation. Figure 11 shows how Sequence ID No. 1 attenuates the repression of IL-10 gene expression associated with dendritic cell maturation after LPS / IFNγ stimulation.

[0124] method CD14+ monocytes were isolated from the blood of three healthy individuals and differentiated into immature dendritic cells (iDCs) by treating them with + / - SEQ ID NO: 1 (400 nM, 0.04 nM) for 5 days with GM-CSF and IL4. The phenotype of the iDCs was confirmed by flow cytometry, and they were further differentiated into mature dendritic cells by treatment with LPS (1 μg / ml) and IFN-γ (IU / ml).

[0125] A total of 12 samples from all three participants (blank; solvent + LPS / IFNγ; SEQ ID NO: 1 400 ng / mL + LPS / IFNγ; SEQ ID NO: 1 0.04 ng / mL + LPS / IFNγ) were subjected to the Human Dendritic Cell & Antigen Presenting Cell RT2 Profiler Array (APC Array) to search for changes in >85 related genes.

[0126] Results and conclusions LPS / IFNγ treatment suppressed the expression of several genes, particularly IL-10. Treatment with 400 ng / ml of SEQ ID NO: 1 significantly reduced the LPS / IFNγ suppression of the IL-10 gene (Figure 11).

[0127] Sequence List SEQUENCE LISTING <110> Revolo Biotherapeutics Limited <120> Method for the treatment of a relapsing-remitting condition <130> PA24-340 <140> JP2024-098776 <141> 2018-01-11 <150> GB 1700555.4 <151> 2017-01-12 <160> twenty five <170> PatentIn version 3.5 <210> 1 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> 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> 16 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <400> 11 Asp Gly Ser Val Val Val Asn Lys Val Ser Glu Leu Pro Ala Gly His 1 5 10 15 <210> 12 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <400> 12 Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp Leu Ala Ala Asp 1 5 10 15 <210> 13 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <400> 13 Asp Gly Ser Val Val Val Asn Lys Val Ser 1 5 10 <210> 14 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <400> 14 Asn Thr Leu Ser Tyr Gly Asp Leu Ala Ala Asp 1 5 10 <210> 15 <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> 15 Xaa His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp 1 5 10 <210> 16 <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> 16 Xaa Asp Gly Tyr Ser Leu Thr Asn Val Asn Leu Gly His 1 5 10 <210> 17 <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> 17 Xaa His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp 1 5 10 <210> 18 <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> 18 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> (12)..(12) <223> D-amino acid residue <220> <221> MISC_FEATURE <222> (12)..(12) <223> NH2 group <400> 19 His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp 1 5 10 <210> 20 <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> 20 His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp 1 5 10 <210> 21 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide molecule <220> <221> MISC_FEATURE <222> (12)..(12) <223> NH2 group <400> 21 His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp 1 5 10 <210> 22 <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> 22 Xaa His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp 1 5 10 <210> 23 <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> 23 Asp Gly Tyr Ser Leu Thr Asn Val Asn Leu Gly His 1 5 10 <210> 24 <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> 24 His Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp 1 5 10 <210> 25 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence inverted for peptide molecule <400> 25 Gly Leu Asn Val Asn Thr Leu Ser Tyr Gly Asp 1 5 10

Claims

1. A pharmaceutical composition for inducing and maintaining therapeutic remission beyond the pharmacokinetic range for an allergic disease, wherein the composition comprises an effective amount The composition comprises a peptide molecule having the amino acid sequence DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 1).

2. A pharmaceutical composition according to claim 1, which causes disease modification.

3. The pharmaceutical composition according to claim 1 or 2, wherein remission is maintained without the need to administer additional peptide doses.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein remission includes a reduction, alleviation, or disappearance of one or more symptoms of an allergic disease.

5. A pharmaceutical composition according to any one of claims 1 to 4, wherein remission includes a reduction, alleviation, or disappearance of one or more symptoms of an allergic disease, with remission lasting for a period significantly longer than the pharmacokinetic half-life of the peptide in the blood.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein remission is maintained when the peptide concentration in plasma is below the lower limit of quantification, and the blood concentration is below the lower limit of quantification of less than 30 ng / mL.

7. The pharmaceutical composition according to claim 6, wherein the peptide concentration in plasma is below the lower limit of quantification, which is a blood concentration of less than 20 ng / mL.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the period of remission of an allergic disease is at least 7 days after the concentration of peptide molecules in the subject's plasma falls below the lower limit of quantification, and the plasma peptide concentration is below the lower limit of quantification, which is a blood concentration of less than 30 ng / mL.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the period of remission of the allergic disease is at least 7 days, 14 days, 28 days, or 6 months after the final administration of the peptide.

10. A pharmaceutical composition according to any one of claims 1 to 9, wherein a single dose of the peptide is administered to a human subject.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the allergic disease is an allergic inflammatory disease.

12. The pharmaceutical composition according to claim 11, wherein the allergic inflammatory disease is atopic dermatitis or rhinitis.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the allergic disease is accompanied by eosinophilia and / or neutrophilia.

14. The pharmaceutical composition according to claim 13, wherein remission comprises a significant decrease in the number of neutrophils and / or eosinophils transported to the site of inflammation in a human subject compared to a control subject that was not administered the peptide molecule.

15. The pharmaceutical composition according to claim 13, wherein remission includes a significant decrease in the number of neutrophils found at the site of inflammation in a human subject compared to a control subject.

16. The pharmaceutical composition according to claim 13 or 14, wherein the allergic disease is a lung disease, and remission comprises a significant decrease in the number of neutrophils and / or eosinophils, which are mobilized to the lungs or found in the circulatory system.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein remission comprises a significant decrease in the number of lymphocytes or a significant increase in the number of macrophages in a human subject compared to a control subject.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein remission comprises a significant change in the amount of one or more inflammatory markers, such as cytokines including IL-4, IL-5, IL-10, or IL-13, in a human subject compared to a control subject.

19. The pharmaceutical composition according to any one of claims 1 to 18, wherein remission includes a significant increase in the amount of IL-10 in human subjects compared to control subjects.

20. The pharmaceutical composition according to any one of claims 1 to 19, wherein remission comprises a significant decrease in the amount of IL-4, IL-5, or IL-13 in a human subject compared to a control subject.

21. The pharmaceutical composition according to any one of claims 1 to 20, wherein the recurrence of the disease includes an increase in the number or severity of symptoms associated with the disease.

22. A pharmaceutical composition comprising a peptide molecule contained in any one of claims 1 to 21, and one or more pharmaceutically acceptable additives, for use in the uses described in any one of claims 1 to 21.

23. The pharmaceutical composition according to any one of claims 1 to 22, wherein the patient is further administered one or more therapeutic agents, or the peptide is given in combination with one or more therapeutic agents.

24. The pharmaceutical composition according to claim 23, wherein the therapeutic agent is selected from disease-modifying agents, analgesics, anti-inflammatory agents, anti-allergic agents, allergen immunotherapy agents, antiviral agents, antibiotics, antibodies, and steroids.

25. The pharmaceutical composition according to claim 23, wherein the therapeutic agent is a bronchodilator.

26. The pharmaceutical composition according to claim 23, wherein the therapeutic agent is selected from adrenocortical steroids, anti-leukotriene agents, cytokines, monoclonal antibodies, and theophylline.

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