WO2007091240A2 - Procédé de traitement de maladies immunologiques - Google Patents

Procédé de traitement de maladies immunologiques Download PDF

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Publication number
WO2007091240A2
WO2007091240A2 PCT/IL2007/000120 IL2007000120W WO2007091240A2 WO 2007091240 A2 WO2007091240 A2 WO 2007091240A2 IL 2007000120 W IL2007000120 W IL 2007000120W WO 2007091240 A2 WO2007091240 A2 WO 2007091240A2
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disease
seq
amino acid
isolated polypeptide
syndrome
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PCT/IL2007/000120
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English (en)
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WO2007091240A3 (fr
Inventor
Yoram Devary
Uziel Sandler
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Immune System Key Ltd.
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Publication of WO2007091240A2 publication Critical patent/WO2007091240A2/fr
Publication of WO2007091240A3 publication Critical patent/WO2007091240A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to the treatment of diseases having an immunological component.
  • CD4+ CD25+ cell populations in vivo and ameliorates type I diabetes in nonobese diabetic mice in a dose-dependent fashion, J. Immun. 171:2270- 2278.
  • InterleuMn-4 Prevent Diabetes in Nonobese Diabetic Mice with Advanced Insulitis, Human Gene Therapy, Vol. 14, No. 1 : 13-23.
  • Cottard, V. et al (2000) Adeno-associated virus-mediated delivery ofIL-4 prevents collagen-induced arthritis, Gene Therapy Vol. 7(22): 1930-1939.
  • Interleukin-10 is a homodimeric cytokine which has potent anti- inflammatory and immunosuppressive effects, especially on monocytes, dendritic cells and macrophages (1) .
  • a single, systemic administration of an adenoviral vector encoding viral IL-10 was found to inhibit murine collagen-induced arthritis (2) .
  • Type I diabetes was prevented in nonobese diabetic mice by early systemic treatment with high doses of a vector expressing murine IL-10 (3) .
  • IL-10 expression will have an effect on those diseases that have an immunological component or etiology such as infectious diseases, acute and chronic inflammatory diseases, cancer, transplantation and autoimmune diseases .
  • autoimmune diseases are mediated by primarily cellular or humoral immune components.
  • autoimmune diseases mediated primarily by cellular components are multiple sclerosis (MS), autoimmune uveitis, autoimmune uveoretinitis, autoimmune thyroiditis, Hashimoto's disease, insulitis, Sjogren's syndrome, experimental autoimmune myocarditis, rheumatoid arthritis (RA), inflammatory bowel disease (IBD), Crohn's disease, and diabetes type I.
  • MS multiple sclerosis
  • autoimmune uveitis autoimmune uveoretinitis
  • autoimmune thyroiditis Hashimoto's disease
  • insulitis insulitis
  • Sjogren's syndrome experimental autoimmune myocarditis
  • RA rheumatoid arthritis
  • IBD inflammatory bowel disease
  • Crohn's disease and diabetes type I.
  • ThI cells secrete the cytokines interleukin 2 (IL-2), interferon- ⁇ (IFN- ⁇ ) and tumor necrosis factor ⁇ (TNF ⁇ ), and induce the cell-mediated (delayed-type hypersensitivity) immune response.
  • Th2 cells secrete the cytokines IL- l ⁇ , IL-4, IL-5 and IL-10, and induce the humoral (antibody) immune response.
  • IL-10 inhibits antigen-presenting cells (APC) such as macrophages which activate ThI cells, while IFN- ⁇ inhibits the production of cytokines by Th2 cells, hi particular, IL-10 has an immunosuppressive effect and inhibits the production of pro-inflammatory mediators involved in cell-mediated autoimmune disease (2) .
  • IL-4 has also been used to treat autoimmune diseases (7)(8)(9) .
  • the peptide encoded by the cDNA has been named TlOl.
  • the peptide is described in copending Intemational Patent Application No. WO 2006/046239 (6) , filed October 26, 2005, whose entire contents are incorporated by reference.
  • a method for treating or preventing a disease capable of being alleviated by interleukin 10 (IL-10) and/or by IL- 4 in a subject in need comprising administering to the subject a therapeutically effective amount of an isolated polypeptide from the group consisting of: (a) an isolated polypeptide comprising an amino acid sequence of SEQ. ID.
  • a disease which may be alleviated by IL-10 and/or IL-4" is used herein to denote diseases having an immunological component or etiology which may be treated either by administration of IL-10 and/or of IL-4 or by inducing the endogenous production of IL-10 and/or of IL-4.
  • diseases include infectious diseases, acute and chronic inflammatory diseases, cancer diseases, transplantation rejection and autoimmune diseases.
  • Infectious diseases which may be treated in accordance with the invention include diseases resulting from bacterial, fungal or protozoan infections.
  • inflammatory diseases which may be treated in accordance with the invention include sepsis, endotoxemia, pancreatitis, uveitis, hepatitis, peritonitis, keratitis, SIRS and injury-induced inflammation.
  • sepsis endotoxemia
  • pancreatitis pancreatitis
  • uveitis hepatitis
  • peritonitis keratitis
  • SIRS injury-induced inflammation
  • autoimmune diseases which may be treated in accordance with the invention include multiple sclerosis (MS), autoimmune uveitis, autoimmune uveoretinitis, autoimmune thyroiditis, Hashimoto's disease, insulitis, Sjogren's syndrome, spontaneous abortions, experimental autoimmune myocarditis, rheumatoid arthritis (RA), inflammatory bowel disease (IBD), Crohn's disease, lupus (SLE), psoriasis and diabetes, particularly type I.
  • MS multiple sclerosis
  • autoimmune uveitis autoimmune uveoretinitis
  • autoimmune thyroiditis Hashimoto's disease
  • insulitis insulitis
  • Sjogren's syndrome spontaneous abortions
  • experimental autoimmune myocarditis rheumatoid arthritis
  • IBD inflammatory bowel disease
  • SLE lupus
  • psoriasis and diabetes, particularly type I.
  • autoimmune diseases which may be treated in accordance with the invention include Acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, Agammaglobulinemia, Allergic asthma, Allergic rhinitis, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome (APS), Autoimmune aplastic anemia, Autoimmune dysautonomia, Autoimmune hepatitis, Autoimmune hyperlipidemia, Autoimmune immunodeficiency, Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune thrombocytopenic purpura (ATP), Axonal & neuronal neuropathies, BaI' s disease, Behnet's disease, Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiac sprue (nontropical), Chagas' disease
  • amino acid will generally refer to an L- amino acid, unless specifically indicated, otherwise.
  • polypeptide consists of the following sequence (SEQ ID. NO: 1):
  • Tl 01 peptide This polypeptide will be referred to herein as the "Tl 01 peptide " or "TlOF.
  • peptide is used herein to denote a peptide, polypeptide or protein.
  • the peptide may be obtained synthetically, through genetic engineering methods, expression in a host cell, or through any other suitable means.
  • a nucleic acid molecule comprising a sequence encoding for the TlOl peptide includes the following sequence (SEQ. ID. NO: T):
  • the TlOl peptide is included in a larger polypeptide encoded by a cDNA which is 84 amino acids long and includes a signal peptide of 33 amino acids on its N-terminal end.
  • the cDNA sequence (SEQ. ID. NO: 3) and amino acid sequence (SEQ. ID. NO: 4) of this longer peptide are as follows:
  • WERK SEQ. ID. NO: 4
  • the polypeptide of SEQ. ID. NO:3 will be referred to herein as the "full TlOl peptide ".
  • the full TlOl peptide may also be used in the method of the invention.
  • active ingredient may be used at times in the specification to denote the active substance used in the method of the invention, such as TlOl or a derivative thereof.
  • the polypeptide consists of an amino acid sequence of SEQ. ID. NO: 1 or SEQ. ID. NO: 4, in which one or more amino acid residues is added, deleted or replaced, without significantly affecting the biological characteristics of the modified molecule as compared to the unmodified molecule.
  • biological characteristics refers to the peptide's ability to exert at least one of the in vitro or in vivo effects that may be exerted by the TlOl peptide or the full TlOl peptide, including but not limited to the biological activities reported below in the Examples.
  • biological characteristics refers to the property of encoding a peptide having similar biological characteristics to that of the TlOl peptide or the full TlOl peptide, including, in particular: (i) a nucleic acid molecule that has a different sequence to that of SEQ. ID. NO: 2 or SEQ. ID.
  • one or more assays can be carried out, such as for example an in vitro, in vivo or a clinical experiment in which a modified peptide is compared to the corresponding unmodified one (namely that of the TlOl peptide or the full TlOl peptide) that is assayed in parallel; or an experiment in which the modified peptide is assayed to examine whether it has a biological effect similar to that of the unmodified peptide as known from separately conducted experiments.
  • Such an experiment may be carried out, for example, in a manner described in the Examples below.
  • the term "without significantly affecting the biological characteristics of the modified molecule as compared to the unmodified molecule denotes the property of encoding a modified peptide of any of the above characteristics.
  • the polypeptide consists of a peptide comprising a partial contiguous sequence from the TlOl peptide including at least 8 amino acid residues, which contiguous sequence is included as a contiguous sequence in said TlOl peptide.
  • a partial TlOl peptide Such a peptide will be referred to herein as a "partial TlOl peptide”.
  • the polypeptide consists of a partial TlOl peptide that comprises a contiguous sequence of 13 amino acid residues beginning from the N-terminal end of the TlOl peptide (amino acid nos. 39 to 51), as follows:
  • a partial TlOl peptide may be a peptide that includes a contiguous sequence of at least 8, 12, 15, 20, 25, 30, 35, 40 or at least 45 amino acid residues that has a degree of identity to a corresponding sequence of at least 8, 12, 15, 20, 25, 30, 35, 40 or at least 45 amino acid residues included in the TlOl peptide, the degree of identity being at least 70%, preferably at least 80%, more preferably at least 90% and particularly at least 95%.
  • TlOl comprising protein A protein or polypeptide comprising an amino acid sequence of the full TlOl peptide, TlOl peptide, modified peptide or a partial TlOl peptide (such protein or polypeptide will be referred to herein as "TlOl comprising protein") may also be used in the method of the invention.
  • the TlOl comprising protein may, for example, be a fusion protein that comprises the full TlOl peptide, the TlOl peptide, a modified peptide or a partial TlOl peptide; it may be a conjugate of a protein or another peptide or polypeptide with the full TlOl peptide, TlOl peptide, modified peptide or partial TlOl peptide; etc.
  • TlOl peptides which may be used in the method of the invention are as follows:
  • DKQLISKAVWWTFFLPSTLWERK (SEQ. ID. NO: 7)
  • PSTLWERK SEQ. ID. NO: 8
  • SEQ. ID. NO: 6 consists of amino acids 6 to 28 of the TlOl peptide
  • SEQ. ID. NO: 7 consists of amino acids 29 to 51 of the TlOl peptide
  • SEQ. ID. NO: 8 consists of amino acids 44 to 51 of the TlOl peptide
  • SEQ. ID. NO: 9 consists of amino acids 36 to 48 of the TlOl peptide
  • SEQ. ID. NO: 10 consists of amino acids 36 to 51 of the TlOl peptide in the reverse order
  • SEQ. ID. NO: 11 consists of amino acids 39 to 43 of the TlOl peptide.
  • partial TlOl peptides are modified peptides derived from any of the peptides defined above, e.g., modified peptides in which one or more amino acids are replaced by another ammo acid by conservative substitution.
  • conservative substitution refers to the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature.
  • Class I Cys
  • Class II Ser, Thr, Pro, Ala, GIy
  • Class III Asn, Asp, GIn, GIu
  • Class IV His, Arg, Lys
  • Class V He, Leu, VaI, Met
  • Class VI Phe, Tyr, Trp
  • substitution of an Asp for another class III residue such as Asn, GIn, or GIu is a conservative substitution.
  • substitutions are made in the amino acid sequence. In another embodiment, two substitutions are made. In a further embodiment, three substitutions are made. The maximum number of substitutions should not exceed that number of amino acids which leaves at least 70%, desirably at least 80%, preferably at least 90%, most preferably at least 95% of the amino acids in the unsubstitued sequence. By one preferred embodiment, the substitutions which include up to 3, at times up to 6 amino acid residues substituted by others, are conservative substitutions.
  • one or more amino acids of the peptides, proteins or polypeptides of the invention may be replaced by D-amino acids, preferably the corresponding D-amino acids.
  • sequences of the reverse order of the above sequences may also be used in the invention.
  • TlOl peptides of SEQ ID NO: 4 or preferably TlOl peptides of SEQ ID NO: 1 or partial TlOl sequences thereof, modified by one or more conservative substitutions may also be used in the method of the invention.
  • peptides include at least 10, or 15, or 20, or 25, or 30, or 35, or 40 amino acid residues, or the entire sequence of the TlOl peptide having the sequence: AAi,- AA 2 -... -AA 5 I, wherein: AA 1 is selected from leucine, isoleucine, valine and methionine;
  • AA 2 is selected from lysine, arginine and histidine;
  • AA 3 is selected from leucine, isoleucine, valine and methionine;
  • AA 4 is selected from tryptophan, phenylalanine and tyrosine;
  • AA 5 is selected from leucine, isoleucine, valine and methionine;
  • AA 6 is selected from serine, threonine, alanine, glycine and proline;
  • AA 7 is selected from serine, threonine, alanine, glycine and proline;
  • AA 8 is selected from glutamine, glutamic acid, aspartic acid and asparagine;
  • AA 9 is selected from serine, threonine, alanine, glycine and proline;
  • AA 10 is selected from leucine, isoleucine, valine and methionine;
  • AA 11 is selected from glutamine, glutamic acid, aspartic acid and asparagine;
  • AA 12 is selected from serine, threonine, alanine, glycine and proline;
  • AA 13 is selected from serine, threonine, alanine, glycine and proline;
  • AA 14 is selected from serine, threonine, alanine, glycine and proline;
  • AA 15 is selected from serine, threonine, alanine, glycine and proline;
  • AA 16 is selected from lysine, arginine and histidine;
  • AA 17 is selected from glutamine, glutamic acid, aspartic acid and asparagine;
  • AA 18 is selected from leucine, isoleucine, valine and methionine;
  • AA 19 is selected from lysine, arginine and histidine;
  • AA 20 is selected from serine, threonine, alanine, glycine and proline;
  • AA 21 is selected from lysine, arginine and histidine;
  • AA 22 is selected from serine, threonine, alanine, glycine and proline;
  • AA 23 is selected from glutamine, glutamic acid, aspartic acid and asparagine;
  • AA 24 is selected from serine, threonine, alanine, glycine and proline;
  • AA 25 is selected from lysine, arginine and histidine;
  • AA 26 is selected from lysine, arginine and histidine;
  • AA 27 is selected from leucine, isoleucine, valine and methionine;
  • AA 28 is selected from serine, threonine, alanine, glycine and proline;
  • AA 29 is selected from glutamine, glutamic acid, aspartic acid and asparagine;
  • AA 30 is selected from lysine, arginine and histidine;
  • AA 31 is selected from glutamine, glutamic acid, aspartic acid and asparagine;
  • AA 32 is selected from leucine, isoleucine, valine and methionine;
  • AA 33 is selected from leucine, isoleucine, valine and methionine;
  • AA 34 is selected from serine, threonine, alanine, glycine and proline;
  • AA 35 is selected from lysine, arginine and liistidine;
  • AA 36 is selected from serine, threonine, alanine, glycine and proline;
  • AA 37 is selected from leucine, isoleucine, valine and methionine;
  • AA 38 is selected from tryptophan, phenylalanine and tyrosine;
  • AA 39 is selected from tryptophan, phenylalanine and tyrosine;
  • AA 40 is selected from serine, threonine, alanine, glycine and proline;
  • AA 41 is selected from tryptophan, phenylalanine and tyrosine;
  • AA 42 is selected from tryptophan, phenylalanine and tyrosine;
  • AA 43 is selected from leucine, isoleucine, valine and methionine;
  • AA 44 is selected from serine, threonine, alanine, glycine and proline;
  • AA 45 is selected from serine, threonine, alanine, glycine and proline;
  • AA 46 is selected from serine, threonine, alanine, glycine and proline;
  • AA 47 is selected from leucine, isoleucine, valine and methionine ' ;
  • AA 48 is selected from tryptophan, phenylalanine and tyrosine;
  • AA 49 is selected from glutamine, glutamic acid, aspartic acid and asparagine;
  • AA 50 is selected from lysine, arginine and histidine;
  • AA 51 is selected from lysine, arginine and histidine.
  • modified peptides based on the full TlOl peptide, TlOl peptide or partial TlOl peptide including the following subsequences (amino acid numbering based on the TlOl peptide):
  • AA 41 and AA 42 are Class VI amino acids, preferably phenylalanine.
  • AA 40 is a Class II amino acid, preferably threonine;
  • AA 41 and AA 42 are Class VI amino acids, preferably phenylalanine; and AA 43 is a Class
  • V amino acid preferably leucine.
  • VI amino acids preferably tryptophan
  • AA 40 is a Class II amino acid, preferably threonine
  • AA 41 and AA 42 are Class VI amino acids, preferably phenylalanine
  • AA 43 is a Class V amino acid, preferably leucine.
  • the peptides and polypeptides used in the method of the invention may be manufactured by any conventional process such as chemical synthesis and recombinant technology.
  • treating or preventing in the context of the present invention refers to the administering of a therapeutic amount of the polypeptide or composition of the present invention which is effective to ameliorate undesired symptoms associated with a disease, to prevent the manifestation of such symptoms before they occur, to slow down the progression of the disease, slow down the deterioration of symptoms, to enhance the onset of remission period, slow down the irreversible damage caused in the progressive chronic stage of the disease, to delay the onset of said progressive stage, to lessen the severity or cure the disease, to improve survival rate or more rapid recovery, to prevent the disease form occurring, or a combination of two or more of the above.
  • treatment in the context used herein also refers to prevention of the disease from occurring.
  • the treatment (also preventative treatment) regimen and specific composition to be administered will depend on the type of disease to be treated and may be determined by various considerations known to those skilled in the art of medicine, e.g. the physicians
  • the "therapeutically effective amount" for purposes herein is determined by such considerations as may be known in the art.
  • the amount must be effective to achieve the desired therapeutic effect which depends on the type and mode of treatment. As is clear to the artisan, the amount should be effective to obtain the improvement of survival rate, to obtain a more rapid recovery, to obtain the improvement or elimination of symptoms or any other indicators as are selected as appropriate measures by those skilled in the art.
  • an effective amount of the active ingredient may be an amount which reduces the symptoms of the disease or even cures the disease for a limited or extended period of time.
  • a therapeutically effective amount of TlOl is typically administered in a single daily dose, although at times a daily dose may be divided into several doses administered throughout the day or at times several daily doses may be combined into a single dose to be given to the patient once every several days, particularly if administered in a sustained release formulation.
  • the active ingredient may be administered as a non-active substance (e.g. prodrug) and be made active only upon further modif ⁇ cation/s by a natural process at a specific site in the subject.
  • the derivative will be such that the therapeutic functionality of the pharmaceutical composition of the invention is preserved.
  • prodrugs are also encompassed by the term "active ingi-edient" as used herein.
  • the method of the invention may also include the administration of drugs in addition to the isolated polypeptide.
  • glucocorticoid drugs may be administered before, together with or subsequently to the administration of the isolated polypeptide.
  • Non-limiting examples of glucocorticoid drugs include dexamethasone,
  • Cortisol cortisone, prednisolone and prednisone.
  • a further embodiment of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of an isolated polypeptide from the group consisting of: (a) an isolated polypeptide comprising an amino acid sequence of SEQ. ID.
  • an isolated polypeptide comprising a contiguous sequence of 13 amino acid residues beginning from the N-terminal of SEQ. ID. NO: 1 ; for use in a method for treating or preventing a disease capable of being alleviated by interleukin 10 (IL-IO) and/or IL-4 in a subject in need.
  • IL-IO interleukin 10
  • IL-4 interleukin 4
  • the administration of the polypeptide to a patient may be together with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier any one of inert, non-toxic materials, which do not react with the active ingredient.
  • the carrier is selected at times based on the desired form of the formulation.
  • the carrier may also at times have the effect of the improving the delivery or penetration of the active ingredient to the target tissue, for improving the stability of the drug, for slowing clearance rates, for imparting slow release properties, for reducing undesired side effects etc.
  • the carrier may also be a substance that stabilizes the formulation (e.g. a preservative), for providing the formulation with an edible flavor, etc.
  • the carriers may be any of those conventionally used and is limited only by chemical-physical considerations, such as solubility and lack of reactivity with the polypeptide, and by the route of administration.
  • the carrier may include additives, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers.
  • the carrier may be an adjuvant, which, by definition are substances affecting the action of the active ingredient in a predictable way.
  • Typical examples of carriers include (a) liquid solutions, where an effective amount of the active substance is dissolved in diluents, such as water, saline, natural juices, alcohols, syrups, etc.; (b) capsules (e.g.
  • the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers), tablets, lozenges (wherein the active substance is in a flavor, such as sucrose and acacia or tragacanth or the active substance is in an inert base, such as gelatin and glycerin), and troches, each containing a predetermined amount of active agent as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; (e) suitable emulsions; (f) liposome formulation; and others.
  • a unit dosage injectable form solution, suspension, emulsion).
  • the pharmaceutical formulation suitable for injection includes sterile aqueous solutions or dispersions and sterile powders for reconstirution into sterile injectable solutions or dispersions.
  • the carrier employed can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, lipid polyethylene glycol and the like), suitable mixtures thereof and vegetable oils.
  • compositions of the invention are administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient's age, sex, body weight and other factors known to medical practitioners.
  • the composition of the invention may be administered in various ways. It can be administered orally, subcutaneously or parenterally including intravenous, intraarterial, intramuscular, intraperitoneally or by intranasal administration, as well as by intrathecal and infusion techniques known to the man versed in the art.
  • a pharmaceutical composition for oral administration comprises an isolated polypeptide in which one or more of the L-amino acids are replaced by the corresponding D-amino acids.
  • a treatment course in humans is usually longer than in animals, e.g. mice, as exemplified herein.
  • the treatment has a length proportional to the length of the disease process and active agent effectiveness.
  • the therapeutic regimen may involve single doses or multiple doses over a period of several days or more.
  • the treatment generally has a length contingent with the course of the disease process, active agent effectiveness and the patient species being treated.
  • Non-aqueous vehicles such as cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and ester, such as isopropyl myristate, may also at times be used as solvent systems for the active ingredient.
  • compositions which enhance the stability, sterility and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents and buffers can be added.
  • antimicrobial preservatives including antimicrobial preservatives, antioxidants, chelating agents and buffers
  • buffers can be added.
  • antibacterial and antifungal agents for example, parabens, chlorobutanol, phenol, sorbic acid and the like.
  • the active ingredient may be formulated in the form of tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like, are usable and may be obtained by techniques well known to the pharmacists.
  • a still further embodiment of the invention relates to a use of an effective amount of an isolated polypeptide from the group consisting of:
  • an isolated polypeptide comprising an amino acid sequence of SEQ. ID. NO: 1, in which one or more amino acid residues is added, deleted or replaced, without significantly affecting the biological characteristics of the modified molecule as compared to the unmodified molecule;
  • an isolated polypeptide comprising a partial contiguous sequence from SEQ. ID. NO: 1 that includes at least 8 amino acid residues, which contiguous sequence is included as a contiguous sequence in said SEQ. ID. NO: 1 ;
  • Another aspect of the invention relates to a method for treating a subject in need by modulating the immunological stimulating activity of dendritic cells (DC) of the subject, comprising:
  • an isolated polypeptide comprising an amino acid sequence of SEQ. ID. NO: I 5 in which one or more amino acid residues is added, deleted or replaced, without significantly affecting the biological characteristics of the modified molecule as compared to the unmodified molecule; iii) an isolated polypeptide comprising a partial contiguous sequence from SEQ. ID. NO: 1 that includes at least 8 amino acid residues, which contiguous sequence is included as a contiguous sequence in said SEQ. ID. NO: 1; and iv) an isolated polypeptide comprising a contiguous sequence of 13 amino acid residues beginning from the N-terminal of SEQ. ID. NO: 1, and
  • Still another aspect of the invention relates to a method of inducing apoptosis in inflammatory cells and/or in cells involved in an autoimmune disease, comprising administering to the subject a therapeutically effective amount of an isolated polypeptide from the group consisting of:
  • apoptosis is induced directly in the inflammatory cells by the polypeptide, and not necessarily through induction of IL-10 or IL-4.
  • Inflammatory cells are cells involved in the pathology of inflammatory diseases such as sepsis, endotoxemia, pancreatitis, uveitis, hepatitis, peritonitis, keratitis, SIRS and injury-induced inflammation.
  • Cells involved in an autoimmune disease are cells which contribute to the development or pathology of an autoimmune disease. A non- exclusive list of autoimmune diseases appears above.
  • Figs. IA, IB, 1C, ID and IE show bar graphs which indicate the levels of various cytokines in human peripheral blood lymphocytes (PBLs) treated with 1 ⁇ g/ml TlOl peptide for 48 hours. The amounts are based on RNA array analysis of PCR gel bands.
  • Fig. IA shows the level of interferon- ⁇ (INF- ⁇ ) in the absence (Control) or presence (TlOl) of TlOl.
  • Fig. IB shows the level of Interleukin 10 (IL-10)
  • Fig. 1C shows the level of IL-I ⁇ receptor
  • Fig. ID shows the level of IL-4
  • Fig. IE shows the level of IL-I ⁇ ;
  • Figs. 2 A and 2B show bar graphs which indicate the levels of INF- ⁇ and IL-10 in mouse splenocytes treated with either TlOl peptide (Fig. 2A) or 13aa TlOl peptide (Fig. 2B), or saline (Control);
  • Fig. 3 is a bar graph showing IL-10 levels in mice injected with TlOl in saline as compared to a control of saline injections alone (Con) every other day.
  • One experimental group received TlOl peptide injection once every other day (1/2), a second group once daily (1/1) and a third group twice daily (2/1);
  • Fig. 4 is a bar graph showing the mean colon length of mice in which IBD was induced by receiving 5% DSS through the drinking water.
  • Three groups of mice were tested: one group received no DSS and no treatment (water); a second group received DSS in the drinking water and was treated with saline injection (DSS w saline); the third group received DSS in the drinking water and was treated with injections of saline that contained TlOl (DSS w TlOl); •
  • Fig. 5 is a graph showing the change in weight (gr) over time (days) of the groups of mice described in Fig. 4;
  • Figs. 6A, 6B and 6C show bar graphs indicating the relative amounts of interleukin-specific cDNA obtained from RNA extracted from colon lymph nodes of the mice described in Fig. 4.
  • Fig. 6 A shows the levels of INF- ⁇ RNA
  • Fig. 6B shows the levels of IL-4 RNA
  • Fig. 6C shows the levels of IL-10 RNA;
  • Figs. 7A, 7B, 7C and 7D show a FACS analysis of mouse splenocytes. The cells were detected for the presence of either FITC (FLl-H) or PE (FL2-H).
  • Fig. 7A shows the analysis of unlabelled cells (control);
  • Fig. 7B shows the analysis of cells labeled with FITC-a ⁇ ti-TlOl;
  • Fig. 7C shows the analysis of cells labeled with PE-anti-mouse CDl Ic;
  • Fig. 7D shows the analysis of cells labeled with both PE-anti-mouse CDl Ic and FITC-anti-TlOl;
  • Fig. 8 shows a bar graph illustrating the effect of TlOl on the level of TGF- ⁇ in mice with IBD, as described in Fig. 4;
  • Fig. 9 shows a bar graph showing the mean colon length of mice treated as described in Fig. 4.
  • the darker (left-hand) bars show results from mice injected with TlOl/saline simultaneously with receiving the DSS 5 while the lighter (right-hand) bars show results from mice injected with TlOl/saline after signs of IBD appeared;
  • Figs. 1OA and 1OB shows pictures of histological sections of colon taken from the mice of Fig. 9 who received saline (A) or TlOl (B); and
  • Figs. HA, HB and HC are graphs showing the results of the treatment of CIA mice with saline (o) or TlOl ( ⁇ ).
  • Fig. HA shows the change in the Arthritics Index as a function of days after immunization
  • Fig. HB shows the change in paw thickness (x 0.01mm)
  • Fig. HC shows the change in the number of arthritic paws.
  • TlOl and derivatives thereof were obtained from Anaspec, Inc. (California). Each peptide has greater than 95% purity. Purification of total RNA from cells was carried out using the Promega SV RNA isolation kit.
  • RT-PCR (using a GHM0055 kit from Biosource, Inc. (USA)) was carried out using specific oligos for the different cytokines.
  • a typical RT-PCR procedure was as follows: 3 microliters from each of the cDNA, 5 microliters of the specific oligos (total), 20 microliters of Readymix Taq polymerase and 17 microliters of DDW, were used.
  • Example 1 The effect of human TlOl peptide (SEQ ID NO:1) on the production of cytokines by human peripheral blood lymphocytes (PBL) was investigated.
  • Human PBLs were fractionated from blood using a Ficoll-Hypaque gradient and incubated at a cell concentration of 10 6 cells/ml for 48 hours at 37 0 C with 1 ⁇ g/ml or no (control) TlOl in RPMI medium + 10% FCS. Total RNA was harvested and cDNA was prepared. The levels of IL- l ⁇ , IL-4, IL-10 and IL- l ⁇ receptor were measured by RT- PCR.
  • the levels of IL-l ⁇ , IL-l ⁇ receptor, IL-4 and IL-IO significantly increased as a result of incubation with TlOl, while the level of INF- ⁇ slightly decreased.
  • the increase ranged between a 2X increase (IL-4) to a 4.7X increase (IL-10).
  • IL-4 2X increase
  • IL-10 4.7X increase
  • the TlOl peptide specifically activates Th2 cells (which secrete IL-l ⁇ , IL-4 and IL-10) and does not affect or inhibits ThI cells (which secrete INF - ⁇ ).
  • Example 2 In this example, the effect of human TlOl peptide on the production of cytokines by mouse spleenocytes was investigated.
  • Mouse spleenocytes at a cell concentration of 10 6 cells/ml were incubated for 24 hours with 0.1 ⁇ g/ml or no (control) TlOl peptide or human partial 13aa TlOl peptide (SEQ ID NO: 5) under the conditions of Example 1.
  • Total RNA was harvested and cDNA was prepared. The levels of IL-10 and INF- ⁇ were measured by RT-PCR.
  • the level of IL-10 was assayed in. three groups of 3 mice each: (1) one group of mice was injected with TlOl twice a day (2/1); (2) another group was injected with the same concentration of TlOl once a day (1/1); (3) the third group of mice was injected once every other day (1/2). All injected doses were the same (72 micrograms/Kg weight). Mice injected with saline once every other day served as control (Con). The results are shown in Fig. 3.
  • the level of IL-10 was increased in all groups following the injection of TlOl peptide to these mice.
  • the best regimen of injections was twice a day.
  • mice Twenty nine-week-old male Balb/c mice (21-25 grams) received water or water containing dextran sulfate (DSS) (5%) for 9 days. DSS induces a Crohn' s-like disease (IBD - inflammatory bowel disease) in mice. During that time the mice were injected every other day with either saline or saline containing TlOl. The body weight of the mice was followed. At the end of the experiment the mice were sacrificed, the colon removed and its length measured.
  • DSS dextran sulfate
  • mice were divided into 3 groups: Group #1 (4 mice) - Control - received normal drinking water (with no DSS) and no treatment;
  • mice 8 mice
  • DSS w saline
  • mice received drinking water that contained 5% DSS and were injected i.p. with TlOl (at a dose of 60 ⁇ g/Kg).
  • the results are shown in Fig. 6.
  • Fig. 6 A it may be seen that the TlOl peptide had no effect on IFN- ⁇ levels in the colon lymph nodes.
  • the level of IL-4 was significantly increased in the group of mice treated with TlOl peptide (Fig. 6B).
  • the results are especially striking with respect to IL-10 as shown in Fig. 6C. It can be seen that the colon lymph nodes of the diseased mice (Group #2) had no IL-10 at all. However, after treatment with the TlOl peptide, the level of IL-10 increased to double the level in the control group.
  • TlOl peptide significantly increases the endogenous IL-10 level, thereby bringing about an alleviation of autoimmune disease symptoms.
  • DC Dendritic cells
  • APC antigen binding cells
  • Fig. 7C shows the CDlIc antigen, indicating the presence of DC.
  • Fig. 7B it may be seen that some of the splenocytes have TlOl receptors.
  • Fig. 7D shows that some of the cells which have a TlOl receptor are DC. This indicates that TlOl is involved in immune response initiated by DC.
  • Th-3-type cells are a unique T-cell subset which primarily secrete the factor TGF- ⁇ , provide help for IgA and have suppressive properties for Th-I and other immune cells.
  • TlOl the level of TGF- ⁇ was determined in the tissue described in Example 5. The results are shown in Fig. 8. It may be seen that TlOl stimulates the amount of TGF- ⁇ in the diseased tissue, indicating another mechanism of suppressing Th-I -mediated inflammatory response.
  • mice were divided into 2 groups. In one group (15 mice), the mice were injected three times a week with TlOl/saline simultaneously with receiving the DSS for eight days, while in the second group (13 mice), the mice were injected with TlOl/saline after signs of IBD appeared once every other day.
  • the mice were sacrificed, the colons removed, and the colon lengths were measured and averaged. The results are presented in Fig. 9.
  • TlOl peptide caused a statistically significant increase in mean colon length as compared to the diseased mice who received saline. It appears that there is no significant difference between treating the mice before or after appearance of the disease symptoms.
  • FIG. 10(A) Histological sections of the colons were taken from diseased mice who were treated with saline or TlOl, and are shown in Fig. 10.
  • Fig. 10(A) the colon structure is degraded and the colon is exsanguinated
  • Fig. 10(B) the colon shows a healthy structure and is provided with blood vessels.
  • Collagen-induced arthritis is a mouse model for human rheumatoid arthritis.
  • TlOl was injected ip into the mice 4 days after LPS injection.
  • the average of macroscopic score was expressed as a cumulative value for all paws, with a maximum possible score of 16 per mouse.
  • the thickness of each paw was also measured with a caliper.
  • the paw swelling for each mouse was calculated by adding the thicknesses of all four paws.
  • the numbers of arthritic paws per mouse were added to represent the number of arthritic paws in a group, with a maximum possible score of 80 per group of 20 mice. The results are presented in Fig. 11.
  • Fig. HA shows the Arthritics Index.
  • Fig. HB shows the degree of paw swelling. The thickness of each paw was also evaluated with spring-loaded caliper. The paw's swelling for each mouse was calculated by adding the thickness of the individual paws (pO.OOOl).
  • Fig. HC shows the number of arthritic paws. The arthritics paws of individual mice were counted and totaled to represent the number of arthritic paws in each experimental group (maximum 80, pO.OOOl).
  • TlOl significantly improves the wellbeing of the CIA mice, and indicate that TlOl can be an effective therapeutic agent for the treatment of rheumatoid arthritis and other autoimmune diseases.

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Abstract

La présente invention concerne un procédé permettant le traitement ou la prévention d'une maladie capable d'être soulagée par l'interleukine-10 (IL-10) et/ou IL-4 chez un sujet qui en a besoin, comprenant l'administration au sujet d'une quantité thérapeutiquement efficace d'un polypeptide de thymus. Des exemples de telles maladies comprennent des maladies infectieuses, des maladies aiguës et chroniques, le cancer, le rejet de greffe et des maladies auto-immunes. L'invention concerne également une composition pharmaceutique comportant le polypeptide de thymus pour le traitement de ces maladies.
PCT/IL2007/000120 2006-02-06 2007-01-31 Procédé de traitement de maladies immunologiques WO2007091240A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008075349A1 (fr) * 2006-12-18 2008-06-26 Immune System Key Ltd. Procédés thérapeutiques utilisant un peptide du thymus
WO2010011315A3 (fr) * 2008-07-25 2010-05-20 Viral Genetics, Inc. Protéines pour utilisation dans le diagnostic et le traitement d'une infection et d'une maladie
US8957031B2 (en) 2007-10-23 2015-02-17 Regents Of The University Of Colorado, A Body Corporate Competitive inhibitors of invariant chain expression and/or ectopic clip binding
WO2015083167A1 (fr) 2013-12-05 2015-06-11 Immune System Key Ltd. Compositions pharmaceutiques et méthodes destinées au traitement et à la prévention du cancer métastatique
WO2017134668A1 (fr) 2016-02-04 2017-08-10 Immune System Key Ltd. Stress du réticulum endoplasmique comme outil prédictif en thérapie anticancéreuse et thérapie combinée pour le traitement du cancer

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006046239A2 (fr) * 2004-10-25 2006-05-04 Immune System Key Ltd Proteine specifique au thymus

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006046239A2 (fr) * 2004-10-25 2006-05-04 Immune System Key Ltd Proteine specifique au thymus

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008075349A1 (fr) * 2006-12-18 2008-06-26 Immune System Key Ltd. Procédés thérapeutiques utilisant un peptide du thymus
US8957031B2 (en) 2007-10-23 2015-02-17 Regents Of The University Of Colorado, A Body Corporate Competitive inhibitors of invariant chain expression and/or ectopic clip binding
US10420813B2 (en) 2007-10-23 2019-09-24 The Regents Of The University Of Colorado, A Body Corporate Competitive inhibitors of invariant chain expression and/or ectopic clip binding
WO2010011315A3 (fr) * 2008-07-25 2010-05-20 Viral Genetics, Inc. Protéines pour utilisation dans le diagnostic et le traitement d'une infection et d'une maladie
WO2015083167A1 (fr) 2013-12-05 2015-06-11 Immune System Key Ltd. Compositions pharmaceutiques et méthodes destinées au traitement et à la prévention du cancer métastatique
US9878015B2 (en) 2013-12-05 2018-01-30 Immune System Key Ltd. Pharmaceutical compositions and methods for the treatment of metastatic cancer
AU2014358671B2 (en) * 2013-12-05 2019-09-12 Immune System Key Ltd. Pharmaceutical compositions and methods for the treatment and prevention of metastatic cancer
WO2017134668A1 (fr) 2016-02-04 2017-08-10 Immune System Key Ltd. Stress du réticulum endoplasmique comme outil prédictif en thérapie anticancéreuse et thérapie combinée pour le traitement du cancer
US10933117B2 (en) 2016-02-04 2021-03-02 Immune System Key Ltd. Endoplasmic reticulum stress as a predictive tool in cancer therapy and a combination therapy for the treatment of cancer
US11813305B2 (en) 2016-02-04 2023-11-14 Immune System Key Ltd. Endoplasmic reticulum stress as a predictive tool in cancer therapy and a combination therapy for the treatment of cancer

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