WO2007122622A1 - Méthode de traitement d'une maladie - Google Patents

Méthode de traitement d'une maladie Download PDF

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Publication number
WO2007122622A1
WO2007122622A1 PCT/IL2007/000506 IL2007000506W WO2007122622A1 WO 2007122622 A1 WO2007122622 A1 WO 2007122622A1 IL 2007000506 W IL2007000506 W IL 2007000506W WO 2007122622 A1 WO2007122622 A1 WO 2007122622A1
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WIPO (PCT)
Prior art keywords
cancer
amino acid
seq
isolated polypeptide
contiguous sequence
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PCT/IL2007/000506
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English (en)
Inventor
Yoram Devary
Uziel Sandler
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Immune System Key Ltd.
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Application filed by Immune System Key Ltd. filed Critical Immune System Key Ltd.
Publication of WO2007122622A1 publication Critical patent/WO2007122622A1/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

Definitions

  • This invention relates to the treatment of various diseases using peptides.
  • Cancer is among the most lethal diseases affecting civilization in general and the Western world in particular. Although billions of dollars have been spent on cancer research, the mortality rate of cancer has not been significantly affected.
  • GM-CSF granulocyte-macrophage-colony stimulating factor
  • the peptide encoded by the cDNA has been named TlOl.
  • the peptide is described in International Patent Application No. WO 2006/046239, filed October 26, 2005, whose entire contents are incorporated by reference. It is important to augment the anti-cancer host response in cancer treatment.
  • TLRs Toll-like receptors
  • TLR9-deficient mice did not show any responses to CpG-DNA, including Th 1 cytokine production and maturation of DCs.
  • a method for preventing and/or treating cancer in a subject in need comprising administrating to the subject a therapeutically effective amount of an isolated polypeptide as defined below.
  • a method for treating diseases which may be alleviated by IFN- ⁇ , GM-CSF and/or IL-2 in a subject in need, comprising administrating to the subject a therapeutically effective amount of an isolated polypeptide as defined below.
  • the disease is cancer.
  • Non-limiting examples of types of cancer which may be treated using the method of the invention include Adrenocortical cancer; Malignant melanoma; Non- melanoma skin cancer; Cutaneous T-cell Lymphoma; Kaposi's Sarcoma; Bladder cancer; Colon cancer; Colorectal cancer; Rectal cancer; Neuroectodermal and Pineal cancer; Childhood Brain Stem Glioma; Childhood Cerebellar Astrocytoma; Childhood Cerebral Astrocytoma; Childhood medulloblastoma; Childhood visual pathway Glioma; Meningioma; Mixed Glioma; Oligodendroglioma; Astrocytoma; Ependymoma; Pituitary adenoma; Metastasic Adenocarcinoma; Acoustic neuroma; Paravertebral Malignant teratoma; Breast cancer; Ductal carcinoma; Mammary gland neoplasia; Ovarian cancer; Carcinoid tumour; Cer
  • Liver cancer Neuroblastoma; Retinoblastoma; Choriocarcinoma; Endocrine cancers; Endometrial cancer; Esophageal cancer; Ewing's Sarcoma; Eye cancer; Gastric cancer;
  • Gastrointestinal cancers Genitourinary cancers; Glioma; Gynaecological cancers; Head and neck cancer; Hepatocellular cancer; Hypopharynx cancer; Islet call cancer; Kidney cancer; Laryngeal cancer; Lung cancer; Lymphoma; Male breast cancer; Melanoma;
  • Sarcoma Skin cancer; Squamous cell carcinoma; Stomach cancer; Testicular cancerthymus cancer; Thyroid cancer; Transitional cells cancer; Trophoblastic cancer;
  • Uterus cancer Acute Lymphatic leukemia; Acute myeloid leukemia; Adenocystic carcinoma; Anal cancer; Bone cancer; Bowel cancer; Ductal carcinoma; Liposarcoma;
  • Neuroblastoma Nephroblastoma and Osteosarcoma.
  • a method for the prevention or treatment of toxic side effects of a drug, particularly a chemotherapeutic drug, or of irradiation in a subject in need comprising administrating to the subject a therapeutically effective amount of an isolated polypeptide as defined below.
  • the toxic side effect is leucopenia or neutropenia.
  • the toxic side effects are treated or prevented by elevating the level of circulating leukocytes in the subject.
  • the isolated polypeptide is administered to the subject together with the drug or irradiation.
  • the drug or irradiation may, for example, be selected from the group consisting of Actinomycin D; Adriamycin; Asparaginase; Bleomycin; Busulphan; Carboplatin;
  • Carmustine Chlorambucil; Cisplatin; Cytaribine; dacarbazine; Daunorubicin;
  • Doxorubicin Prorubicin; Etoposide; Fludarabine; Fluorouracil; Gemcitabine;
  • Treosulfan; Vinblastine; Vincristine; Vindesine; Vinorelbine; radiotherapy and immuno therapy Also included in the invention is the use of a therapeutically effective amount of an isolated polypeptide as defined below in the preparation of a pharmaceutical composition for use in a method according to the invention.
  • composition comprising a therapeutically effective amount of an isolated polypeptide as defined below for use in a method according to the invention.
  • a method for treating a subject in need by modulating the immunological stimulating activity of antigen presenting cells (APC) of the subject particularly dendritic cells and monocytes.
  • the immunological stimulating activity of the APCs may be modulated, for example, by increasing the levels of toll-like receptors (TLR) on the APC cells.
  • TLR toll-like receptors
  • An increase in the activity of the APCs can have an inhibitory effect on cancer cells as well as strengthening the function of the innate immune system against various pathogenic microorganisms such as viruses, bacteria and parasites.
  • this aspect includes a method for treating a subject in need by modulating the immunological stimulating activity of antigen presenting cells (APC) of the subject, comprising:
  • polypeptides of the invention induce the expression of TLR.
  • this aspect of the invention includes several embodiments:
  • the polypeptides of the invention may be administered together with ligands of TLR in order to enhance their adjuvant effect for different purposes such as immunization and as anti cancer treatment.
  • TLR ligands are given below.
  • Ligands of TLR2 include:
  • Heat-Killed Bacteria such as Acholeplasma laidlawii; Listeria monocytogenes; Legionella pneumophila; Porphyromonas gingivalis;
  • Staphylococcus aureus Lipoglycans such as Mycobacterium smegmatis; Lipopolysaccharide such as Porphyromonas gingivalis; Lipoteichoic Acids such as Bacillus subtilis; Staphylococcus aureus; Peptidoglycans such as Bacillus subtilis; Escherichia coli 0111:B4; Escherichia coli K12; Staphylococcus aureus;
  • Synthetic Lipoproteins such as FSLl (a diacylated synthetic lipoprotein derived from Mycoplasma salivarium similar to MALP-2); Pam3CSK4 (a synthetic tripalmitoylated lipopeptide (LP) that mimicks the acylated amino terminus of bacterial LPs); Pam2CSK4 (a synthetic diacylated LP).
  • Ligands of TLR3 include Polyinosine-polycytidylic acid (poly(I:C)).
  • Ligands of TLR4 include LPS such as E. coli 0111:B4; E. coli K12; S. Minnesota; msbB E. coli mutant and Lipid A.
  • Ligands of TLR5 include Flagellin such as B. subtilis; S. typhimurium.
  • Ligands of TLR7/8 recognize GU-rich short single-stranded RNA as well as small synthetic molecules such as imiquimod and loxoribine.
  • Ligands of TLR9 recognize specific unmethylated CpG-ODN sequences that distinguish microbial DNA from mammalian DNA.
  • the polypeptides of the invention may be used in immunization as an adjuvant, due to their influence on TLR.
  • the polypeptides of the invention may be used to enhance the innate immune system against various pathogenic microorganisms such as viruses, bacteria and parasites.
  • this aspect of the invention includes a method of immunization comprising administering a vaccine comprising an effective amount of an isolated polypeptide as defined below and a pharmaceutically acceptable carrier.
  • This aspect also includes a method for treating a disease caused by bacteria, viruses, fungi and/or parasites comprising administering to a subject in need a therapeutically effective amount of an isolated polypeptide as defined below and a pharmaceutically acceptable carrier. All of the above aspects of the invention involve the administration to a subject in need, or to his cells, of the polypeptides described below.
  • polypeptide consists of the following sequence (SEQ ID. NO: 1): LHLWLSGEPVQSSGTKDMRSKSDSKRVSDKQLISKAVWWT FFLPSTLWERK (SEQ. ID. NO: 1)
  • TlOl peptide This polypeptide will be referred to herein as the "TlOl peptide " or "TlOl”.
  • NF 40 may also be used to denote the TlOl peptide.
  • 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: 2):
  • 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:
  • MMALRSQGLMLPQSCPQLAFLTLSALAAVSFSALHLWLSG EPVQSSGTKDMRSKSDSKRVSDKQLISKAVWWTFFLPSTL WERK SEQ. ID. NO: 4
  • the polypeptide of SEQ. ID. NO: 4 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 described below in the Examples.
  • biological characteristics with respect to a nucleic acid molecule, 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.
  • modified molecule without significantly affecting the biological characteristics of the modified molecule as compared to the unmodified molecule " means to denote that the modified molecule retains a biological activity qualitatively similar to that of the unmodified molecule. With respect to a modified peptide, this means that it retains one or more of the biological characteristics of a peptide of SEQ. ID. NO: 1 or SEQ. ID. NO: 4, including, among others, its therapeutic utilities, as specified in this specification, as well as its in vitro and in vivo activities reported in the Examples below.
  • 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 experiment.
  • Such an experiment may be carried out, for example, in manner described in the Examples below.
  • amino acid refers to an L-amino acid, unless explicitly indicated otherwise.
  • 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 the TlOl peptide.
  • 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:
  • 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 amino 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. In a further embodiment, one or more amino acids may be replaced by D-amino acids, preferably by the corresponding D-amino acids. In a further embodiment, SEQ ID
  • NO:11 is excluded from its amino acids being replaced by D-amino acids.
  • sequences of the reverse order of the above sequences may also be used in the invention.
  • 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.
  • These 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 51 , 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;
  • AAj8 is selected from leucine, isoleucine, valine and methionine;
  • AA 19 is selected from lysine, arginine and histidine;
  • AA 2O 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 histidine;
  • 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 5O is selected from lysine, arginine and histidine;
  • AA 51 is selected from lysine, arginine and histidine. Included are also 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 38 and AA 39 are Class VI amino acids, preferably tryptophan; AA 40 is a Class II amino acid, preferably threonine; and AA 41 and AA 42 are Class VI amino acids, preferably phenylalanine.
  • AA 38 and AA 39 are Class 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; and
  • 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 modification/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 ingredient" as used herein.
  • the method of the invention may also include the administration of drugs in addition to the isolated polypeptide.
  • chemotherapeutic drugs may be administered before, together with or subsequent to the administration of the isolated polypeptide.
  • chemotherapeutic drugs include busulfan, cisplatin, carboplatin, chlorambucil, cyclophosphamide, ifosfamide, dacarbazine, mechlorethamine (nitrogen mustard), melphalan, temozolomidestreptozocin, carmustine, lomustine, 5-fluorouracil, capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, pemetrexeddaunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantronetopotecan, irinotecanetoposide, teniposidetaxanes (paclitaxel, docetaxel),
  • 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 (d) 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 IFN- ⁇ , GM-CSF and/or IL-2 in a subject in need.
  • the administration of the polypeptide to a patient may be together with a pharmaceutically acceptable carrier.
  • the term “pharmaceutically acceptable carrier” it is meant 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.
  • compositions of the present invention When administering the compositions of the present invention parenterally, it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).
  • the pharmaceutical formulation suitable for injection includes sterile aqueous solutions or dispersions and sterile powders for reconstitution 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.
  • 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 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.
  • Fig. 1 is a bar graph showing BrdU incorporation in human PBL incubated with the TlOl peptide as compared to control (Con).
  • the Y-axis shows the optical density at 450 nm — a higher OD signifies a higher BrdU incorporation;
  • Fig. 2 is a bar graph showing INF- ⁇ serum levels (pg/ml) in mice injected i.p. twice a day for 6 days with 60 ⁇ g TlOl/Kg weight as compared to a saline control. Each group consisted of 6 mice, and the sera of the mice of each group were pooled prior to being assayed;
  • Fig. 3 is a bar graph showing IL-2 serum levels (pg/ml) in the mice described in Fig. 2;
  • Fig. 4 is a bar graph showing cell count per microliter, of different human white blood cell populations - monocytes (MONO), lymphocytes (Lym) and whole white blood cell population (WBC) - following incubation of the cells in either saline containing TlOl peptide (TlOl) or saline alone (Saline). The number of monocytes was divided by 1000 in order to include all of the results in one graph;
  • Fig. 5 is a bar graph showing cell count per microliter of human red blood cells following incubation of the cells in either saline containing TlOl peptide (TlOl) or saline alone (Saline);
  • Fig. 6 is a bar graph showing the fold of increase in tumor size in Balb/C mice induced with mammary carcinoma and treated with either saline with TlOl peptide or with saline alone;
  • Fig. 7 is a graph showing the rate of growth (in %) of the tumor referred to in Fig. 6;
  • Figs. 8, 9 and 10 are bar graphs showing INF- ⁇ , IL-2 and GM-CSF serum levels, respectively, (pg/ml or fold induction) in mice with induced carcinoma by injecting 10 6 EMT carcinoma cells s.c. The mice were treated as in Fig. 2;
  • Figs. 11 and 12 are bar graphs showing fold induction (by PCR) of Toll-like Receptor (TLR) -2 and 4, respectively, in spleenocytes of mice as in Fig. 2; and
  • Fig. 13 is a bar graph showing leukemia tumor size in mice treated with either saline with TlOl peptide or with saline alone.
  • TlOl and derivatives thereof were obtained from Anaspec, Inc. (California). Each peptide has greater than 95% purity.
  • Example 1 The TlOl peptide was tested for its ability to activate proliferation of human peripheral blood lymphocytes (PBL). The proliferation activity was measured by 5- bromo 2'-deoxy-uridine (BrdU) incorporation into these cells.
  • Human PBL were isolated on a Ficole gradient from blood taken from volunteers and then plated in 96-well plates, using RPMI 1640 + 10% FCS + Pen/Str.
  • the cells were treated with 0.1 ⁇ g/well of the TlOl peptide or with saline (Con), and after 48 hours they were labeled for 6 hours with BrdU and tested for BrdU incorporation into their nucleus.
  • TlOl was able to increase BrdU incorporation, signifying that TlOl was able to induce the cells to increase their proliferation rate.
  • mice Two groups of 6 mice each were injected i.p. twice a day for 6 days with either TlOl (60 ⁇ g/Kg weight) or with saline. The serum of the mice of each group was pooled prior to being assayed for INF- ⁇ or IL-2 (pg/ml). Results
  • IFN- ⁇ and IL-2 The levels of IFN- ⁇ and IL-2 are shown in Figs. 2 and 3, respectively. It may be seen that injection of TlOl caused a significant increase hi both cytokines.
  • Example 3 The purpose of the experiment was to examine how the TlOl peptides influence the immune response in mice and whether the TlOl peptide can influence any of the components of the immune system both with respect to the level of different cell types and in the ratio between different subpopulations.
  • TlOl peptide or saline 50 microgram/Kg of TlOl peptide or saline were injected (twice a day for 8 days) into Balb/C mice (10 mice per group). CDC analysis was made of the blood and the percentage of different subpopulations of white blood cells (WBC) (monocytes (MONO), lymphocytes (LYM) and total WBC (WBC)) was analyzed. Platelets and RBCs were used as controls.
  • WBC white blood cells
  • WBC white blood cells
  • LYM lymphocytes
  • WBC total WBC
  • saline As a control, the levels of platelets and red blood cells (RBC) were also examined. The difference between saline and TlOl peptide injection was found to be less than 5%, as shown in Fig. 5 (results not shown for platelets).
  • TlOl may be used to prevent or treat toxic side effects, such as leukopenia, neutropenia and myelosuppression, caused by a drug, particularly a chemotherapeutic drug, or by irradiation.
  • mice Eight week Balb/C mice were injected with mammary carcinoma EMT6/CTX cells, and after the size of the tumor reached 4 mm 2 , the mice were injected with TlOl peptide (50 microgram/Kg) twice a day for 8 days. The size of the tumor was measured every other day as well as at the conclusion of the experiment. Results
  • mice treated with the TlOl peptide the fold increase of the tumor size was 4.3, as compared to the original size of the tumor before injection, hi mice that were treated with saline, the fold increase of the tumor size was 6.7.
  • TlOl was able to significantly reduce tumor growth.
  • the rate of growth of the tumor is shown in Fig. 7. It can be seen that TlOl was able to significantly decrease the growth rate of the tumor.
  • TlOl Toll-like Receptors
  • mice Eight week Balb/C mice were injected with 10 6 EMT carcinoma cells, the mice were injected with TlOl peptide (50 microgram/Kg) twice a day for 8 days.
  • TlOl causes a very significant increase in the levels of INF- ⁇ , IL-2 and GM-CSF, and in the levels of TLR-2 and TLR-4.
  • mice 20 DBA/2 mice were injected sc with 0.8xl0 6 P815 cells (Uyttenhove , C et al. J Exp Med. 1980; 152: 1175-1183), which are mastocytoma cells, being leukemia of mast cells.
  • the mice were divided into 2 equal groups. Group A was injected ip twice a day with TlOl (60 ⁇ g/Kg) and Group B was injected ip twice a day with saline. After 11 days, the size of the tumors was measured. Results
  • TlOl very significantly inhibited the development of the tumor.
  • TlOl is shown to have a broad spectrum effect on various types of cancers.

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Abstract

L'invention concerne une méthode pour prévenir ou traiter un cancer chez un sujet en ayant besoin, consistant à administrer au sujet une quantité thérapeutiquement efficace d'un nouveau polypeptide du thymus, ou un fragment pharmaceutiquement actif de celui-ci. Elle concerne aussi une méthode pour traiter ou prévenir une maladie pouvant être soulagée par l'interféron gamma (IFN-γ) et/ou par l'interleukine 2 (IL-2) et/ou par GM-CSF chez un sujet en ayant besoin, qui consiste à administrer au sujet une quantité thérapeutiquement efficace du polypeptide. Elle concerne de plus une méthode pour prévenir ou traiter des effets secondaires toxiques d'un médicament ou d'une irradiation, qui consiste à administrer à un sujet en ayant besoin une quantité efficace du polypeptide.
PCT/IL2007/000506 2006-04-24 2007-04-23 Méthode de traitement d'une maladie WO2007122622A1 (fr)

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Cited By (3)

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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
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

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WO1998001557A2 (fr) * 1996-07-05 1998-01-15 Schering Corporation Reactifs apparentes a des chemokines mammaliennes
WO2006046239A2 (fr) * 2004-10-25 2006-05-04 Immune System Key Ltd Proteine specifique au thymus

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WO1998001557A2 (fr) * 1996-07-05 1998-01-15 Schering Corporation Reactifs apparentes a des chemokines mammaliennes
WO2006046239A2 (fr) * 2004-10-25 2006-05-04 Immune System Key Ltd Proteine specifique au thymus

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Title
HARRIS C A ET AL: "Three distinct human thymopoietins are derived from alternatively spliced mRNAs", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE, WASHINGTON, DC, US, vol. 91, no. 14, 1994, pages 6283 - 6287, XP002379435, ISSN: 0027-8424 *

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
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
CN105939724A (zh) * 2013-12-05 2016-09-14 免疫系统密钥有限公司 用于治疗和预防转移癌的药物组合物和方法
JP2017505755A (ja) * 2013-12-05 2017-02-23 イミューン システム キー リミテッド 転移がんの治療および予防のための医薬組成物および方法
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
CN105939724B (zh) * 2013-12-05 2020-01-14 免疫系统密钥有限公司 用于治疗和预防转移癌的药物组合物和方法
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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