WO2018016674A1 - Composition pharmaceutique pour le traitement ou la prévention de tumeurs cérébrales, contenant un inhibiteur de midkine - Google Patents

Composition pharmaceutique pour le traitement ou la prévention de tumeurs cérébrales, contenant un inhibiteur de midkine Download PDF

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Publication number
WO2018016674A1
WO2018016674A1 PCT/KR2016/009659 KR2016009659W WO2018016674A1 WO 2018016674 A1 WO2018016674 A1 WO 2018016674A1 KR 2016009659 W KR2016009659 W KR 2016009659W WO 2018016674 A1 WO2018016674 A1 WO 2018016674A1
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midkine
temozolomide
tumor
cells
pharmaceutical composition
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Korean (ko)
Inventor
오진미
남도현
강현주
김세희
한수지
공두식
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Samsung Electronics Co Ltd
Samsung Life Public Welfare Foundation
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Samsung Electronics Co Ltd
Samsung Life Public Welfare Foundation
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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
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4188—1,3-Diazoles condensed with other heterocyclic ring systems, e.g. biotin, sorbinil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • composition for the treatment or prevention of brain tumors containing a midkine inhibitor containing a midkine inhibitor
  • compositions for the treatment and / or prevention of brain tumors comprising a midkine inhibitor or a combination of midkine inhibitors and temozolomide 3 ⁇ 4111020101 ⁇ (16), and the treatment of temozolomide of midkine inhibitors and / or Or use for overcoming resistance to radiation therapy.
  • a brain tumor is a tumor in the brain tissue or the membrane tissue surrounding it.
  • the tumor is a secondary brain tumor that has spread from the primary brain tumor to the brain tissue or the meninges in other parts.
  • Brain tumors found in adults include glioma (Gl ioma), glioblastoma (Gl ioblastoma), meningioma (meningoma), pituitary adenoma (adenomas), and schwannoma.
  • glioblastoma (Gl ioblastoma) is a tumor originating from glial cells of brain tissue, which accounts for 12-15% of all brain tumors, and is the most common primary brain tumor in adults (3.19 / 100, 000; Cancer Epidemiol Biomarkers Prev. 2014 Oct; 23 (10): 1985-96).
  • Glioblastomas unlike other tumors, are densely stretched between cells and tissues and are relatively fast in growth and fast in metastasis. Increasing brain pressure causes severe headaches, nausea and vomiting in the morning. Symptoms include epileptic seizures, memory loss and behavioral changes. Neural function may be reduced due to the tumor itself or brain edema accompanying the tumor, resulting in symptoms such as limb movement or sensory impairment, facial paralysis, speech impairment, cognitive impairment, and left and right disability.
  • Glioblastoma treatment similar to other brain tumors, is mainly performed by surgical removal of the tumor as much as possible, combined with radiation therapy and temodal chemotherapy.
  • the prognosis is around 15 months and the prognosis with a 5 year survival rate of 7% is extremely poor (N Engl J Med 2008; 359: 492-507).
  • glioblastoma and / or conventional therapies eg, temozolomide and / or radiation therapy
  • midkine inhibitors alone or in combination with midkine inhibitors and temozolomide
  • the use of midkine to overcome resistance to is provided.
  • One example provides a pharmaceutical composition for the prevention and / or treatment of brain tumors, including a midkine (Mi dkine) inhibitor as an active ingredient.
  • a pharmaceutical composition for the prevention and / or treatment of brain tumors including a midkine (Mi dkine) inhibitor as an active ingredient.
  • Mi dkine midkine
  • Another example provides a pharmaceutical composition for preventing and / or treating brain tumors comprising a midkine inhibitor and temozolomide as an active ingredient.
  • Other examples include a midcaine inhibitor (or a pharmaceutical composition comprising a midcaine inhibitor as an active ingredient) or a combination of a midcaine inhibitor and temozolomide (or a pharmaceutical composition comprising a midcaine inhibitor and temozolomide as an active ingredient).
  • Another example provides a method of preventing and / or treating a brain tumor, comprising administering a midkine inhibitor to a subject in need of the prevention and / or treatment of a brain tumor.
  • the method of preventing and / or treating the brain tumor may further include irradiating before, simultaneously or after administering the midkine inhibitor.
  • Another example is the prevention of brain tumors with midkine inhibitors and temozolomide.
  • the method of preventing and / or treating the brain tumor may further include irradiating before, simultaneously or after the step of concurrently administering the midkine inhibitor and temozolomide.
  • Brain tumors targeted for the prophylaxis and / or treatment may be glioblastomas or brain tumors (eg, glioblastomas) resistant to existing anticancer therapies such as temozolomide and / or radiation therapy.
  • Another example provides a pharmaceutical composition for inhibiting tumor stem cells of brain tumors, including the midkine inhibitor as an active ingredient, inhibiting stem cell function of tumor cells, or preventing recurrence of brain tumors.
  • Other examples include administering a midkine inhibitor to a subject in need of inhibiting tumor stem cells of a brain tumor or inhibiting stem cellization of tumor cells, inhibiting tumor stem cells of a brain tumor, inhibiting stem cell capacity of tumor cells, or Provides a method for preventing recurrence of brain tumors.
  • midkine inhibitors to overcome resistance to or enhance resistance to existing anticancer therapies, such as anticancer therapies for brain tumors (eg, radiation therapy and / or temozolomide therapy, etc.).
  • one embodiment provides a pharmaceutical composition for overcoming resistance or enhancing resistance to radiation therapy and / or temozolomide containing a midkine inhibitor as an active ingredient.
  • Another example is a method of overcoming resistance to temozolomide and / or radiation treatment comprising administering a midkine inhibitor to a subject in need of overcoming resistance or enhancing resistance to radiation therapy and / or temozolomide, and Provide a method of promoting response.
  • Another example provides the use of a midkine inhibitor for overcoming or enhancing resistance to radiation therapy and / or temozolomide.
  • the resistance to temozolomide and / or radiation treatment may be resistance to temozolomide and / or radiation treatment of brain tumors (eg, glioblastoma). And / or pharmaceutical compositions for enhancing the efficacy of radiation therapy.
  • Another example provides a method of enhancing the efficacy of temozolomide and / or radiation treatment comprising administering a midkine inhibitor to a subject in need of enhancing the efficacy of temozolomide and / or radiation therapy.
  • Another example provides the use of a midcaine inhibitor for enhancing the efficacy of temozolomide and / or radiation therapy.
  • the subject in need of enhancing the efficacy of the temozolomide and / or radiation treatment may be a brain tumor patient, eg, glioblastoma patient, or a brain tumor patient, such as glioblastoma patient, resistant to temozolomide and / or radiation therapy. have.
  • Another example includes contacting a candidate compound with a brain tumor test group cell, and measuring the expression amount (m NA amount) of the midkine protein and / or the midkine gene of the test group cell after contacting the candidate compound, wherein the candidate compound
  • An agent or temozolo for overcoming resistance to temozolomide in brain tumors which comprises comparing the amount of midkine protein and / or the expression of midkine genes in the brain tumor comparison group cells which did not come into contact with the candidate compound or before contact.
  • the candidate compound overcomes the resistance to temozolomide of the brain tumor. It may further comprise the step of selecting as a candidate for the preparation for the therapeutic agent or temozolomide resistant brain tumor.
  • the brain tumor test group cells and brain tumor comparison group cells may be (isolated) from the same brain tumor patient.
  • the brain tumor may be glioblastoma
  • the brain tumor cells may be glioblastoma .cell line.
  • the expression level of midkine increases, and the higher the expression level of midkine, the worse the prognosis of the brain tumor.
  • temozolomide administration and / or radiation treatment which is a typical conventional brain tumor treatment
  • temozolomide administration or radiation therapy alone or temozolomide administration and radiation treatment Compared with the combined case, it was confirmed that a significant brain tumor treatment effect can be obtained and anticancer effect can be obtained even for brain tumors resistant to temozolomide and / or radiation therapy.
  • Midkine (MDK), also called neurite growth-promoting factor 2 (NEGF2), is a protein encoded by the MDK gene.
  • Midcaine is a cell's self-secreting growth factor.
  • Midkine is a low molecular weight heparin-binding growth factor and a nonglycosylated protein consisting of two domains linked by disulfide bridges.
  • the midkine or its subcutaneous gene described herein may be (isolated from mammals) derived from mammals such as humans, primates such as primates, or rodents such as mice and rats.
  • the midkine may be a human midkine (e.g., NCBI Accession Numbers.
  • mRNA human midkine gene
  • mouse midkine gene e.g., NCBI Accession Numbers. .1, ⁇ _001291482.1, ⁇ _001291483.1, etc.
  • the midkine gene is human midkine (eg, NCBI Accession Numbers.
  • Temozolomide 1 ⁇ 21! 1020101 ⁇ (16; TNZ) is also known as Temodar, Temodal, Temcad, etc., and the IUPAC name is 4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo. [4.3.0] nona-2, 7, 9-triene-9-carboxamide (4-methy 1-5-0X0-2,3,4,6,8-pent azab i eye 1 is 4.3.0 ] nona-2, 7, 9-tr i ene—9—car boxam i de).
  • Temozolomide is a kind of alkylating agent and is an anticancer agent widely used in the treatment of brain tumors.
  • the brain tumor refers to all tumors generated in the brain tissue or the membrane tissue surrounding it, and may be a primary brain tumor or a metastatic brain tumor.
  • the brain tumor can be glioma (Gl ioma), glioblastoma (Ml ioblastoma), meningioma (meningoma), pituitary adenoma (adenomas), or schwannomas, for example glioblastoma.
  • One embodiment of the present invention provides a pharmaceutical composition for the prevention and / or treatment of brain tumors comprising a midkine inhibitor as an active ingredient.
  • the midkine inhibitor may be included in a pharmaceutically effective amount to achieve the desired effect.
  • Another example provides a method of preventing and / or treating a brain tumor, comprising administering a midkine inhibitor to a subject in need of the prevention and / or treatment of a brain tumor.
  • the midkine inhibitor can be administered in a pharmaceutically effective amount to achieve the desired effect.
  • the method may further comprise identifying a subject in need of prevention and / or treatment of brain tumors prior to the administering step.
  • tumor cells of brain tumors eg, glioblastoma
  • midkine at the protein and / or gene levels.
  • Effective and fundamental cancer treatment is possible, and it is also excellent in preventing cancer from recurring.
  • temozolomide administration and / or radiation treatment which is a typical conventional brain tumor treatment
  • temozolomide administration or radiation therapy alone when temozolomide administration or radiation therapy alone is performed, or when temozolomide administration and radiation treatment are combined
  • anticancer effect can be obtained for brain tumors resistant to temozolomide and / or radiation therapy, and the concentration of temozolomide and / Alternatively, it is possible to reduce the radiation dose or to increase the administration and / or the interval between irradiation, thereby reducing the side effects appearing in the patient.
  • the prevention and / or treatment of the brain tumor may further comprise a step of administering temozolomide and / or irradiation to the subject to obtain a more elevated effect.
  • a pharmaceutical composition for preventing and / or treating brain tumors comprising a midkine inhibitor and temozolomide as an active ingredient.
  • the midkine inhibitor and temozolomide pharmaceutical composition may be a form for simultaneous administration of two drugs including a single formulation (combinator) in which the midcaine inhibitor and temozolomide are mixed.
  • the midkine inhibitor and temozolomide pharmaceutical composition may be formulated with a midkine inhibitor and temozolomide, respectively, to simultaneously or sequentially administer the two drugs.
  • the pharmaceutical composition comprises a first pharmaceutical composition comprising a midkine inhibitor as an active ingredient and a second pharmaceutical composition comprising temozolomide as an active ingredient, the pharmaceutical composition for concurrent administration for simultaneous or sequential administration Can be.
  • sequential administration the order may be interchanged.
  • kits may include the drug and the irradiation device in one connected state or as a series of treatments and separated from each other so that radiation can be continuously irradiated simultaneously with or without the administration of the drug.
  • the irradiation apparatus can be selected and used among all conventional apparatuses used to irradiate radiation of a desired radiation dose (e.g., radiation dose applied to the treatment of brain tumors).
  • the irradiation device may emit a radiation dose of about lGy to about 6Gy, about lGy to about 4Gy, about lGy to about 2Gy, about 2Gy to about 6Gy, about 2Gy to about 4Gy, or about 4Gy to about 6Gy at a time. It may be possible to investigate, but is not limited thereto.
  • devices that can be used for irradiation include, but are not limited to, IBL 437C blood irradiator (CIS US, Inc., Bedford, MA).
  • midkine inhibitors are administered to patients with brain tumors, such as glioblastoma
  • stem cells of brain tumors eg, glioblastoma
  • brain tumor tumor stem cells are reduced (killing, inhibiting proliferation, etc.).
  • the effects can be obtained, and the recurrence rate of brain tumors can be remarkably reduced by such tumor stem cell inhibition.
  • another example provides a pharmaceutical composition for inhibiting tumor stem cells of brain tumors or inhibiting stem cell capacity of tumor cells comprising a midkine inhibitor as an active ingredient.
  • Other examples include inhibiting tumor stem cells of brain tumors, inhibiting stem cellization of tumor cells, comprising administering a midkine inhibitor to a subject in need of inhibiting tumor stem cells of a brain tumor or inhibiting stem cellization of tumor cells, or Provides a method for preventing brain tumor recurrence.
  • the tumor stem cell inhibition refers to the induction of death, suppression of proliferation, etc. of tumor stem cells of brain tumors (eg, glioblastomas) to be reduced or eliminated numerically or quantitatively.
  • Inhibiting stem cellization means inhibiting stem cell tumor cells of a brain tumor (eg, glioblastoma).
  • Prevention of recurrence of brain tumors means preventing the formation or worsening after treatment or improvement of brain tumors (eg, glioblastoma).
  • Temozolomide can each be included in a pharmaceutically effective amount to achieve the desired pharmacological effect.
  • the method of preventing and / or treating a brain tumor comprising administering the midkine inhibitor to a subject in need of the prevention and / or treatment of a brain tumor, the radiation before, concurrently or after the step of administering the midkine inhibitor It may be to further include the step of examining.
  • Another example provides a method of preventing and / or treating a brain tumor, comprising co-administering a midkine inhibitor and temozolomide to a subject in need of prevention and / or treatment of a brain tumor.
  • the concomitant administration may be performed simultaneously or sequentially in the order of administering the midkine inhibitor and temozolomide (for example, administering the first step including the midkine inhibitor and temozolomide, simultaneously or in any order). May be performed sequentially).
  • the method of preventing and / or treating the brain tumor may further include irradiating before, simultaneously or after the step of concurrently administering the midkine inhibitor and temozolomide.
  • the midkine inhibitor and temozolomide can be administered in a pharmaceutically effective amount, respectively.
  • the method may further comprise identifying a subject in need of prevention and / or treatment of brain tumors prior to the administering step.
  • the cerebral tumor targeted by the prophylaxis and / or treatment may be a brain tumor (eg, glioblastoma) resistant to conventional anticancer treatments such as glioblastoma or temozolomide and / or radiation therapy.
  • the treatment of brain tumors may include the action associated with the reduction or disappearance of tumor cells of brain tumors (inhibition of tumor cell proliferation, apoptosiosis, etc.), and / or inhibition of stem cell capacity of brain tumors and / or prevention of recurrence of brain tumors, and / or existing It can be interpreted as meaning including overcoming resistance to anticancer therapy (eg, radiation therapy and / or temozolomide therapy, etc.) (increasing the therapeutic reaction sensitivity).
  • anticancer therapy eg, radiation therapy and / or temozolomide therapy, etc.
  • the administration and / or radiation subject may be a patient with a brain tumor (e.g. glioblastoma) or a human, monkey, etc. at risk of suffering from a brain tumor (e.g. glioblastoma).
  • Primates including At least one selected from the group consisting of cells and tissues and cultures thereof.
  • the subject may be isolated from mammals, including temozolomide and / or primates including humans, monkeys, etc., innate or acquired (acquired) resistance to radiation therapy, rodents including mice, rats, and the like. Cells and tissues and cultures thereof.
  • midkine inhibitor may be used as a generic term for all compounds and compositions that target and act to decrease and / or lose binding and / or activity by targeting a midkine protein or gene.
  • the midkine inhibitor may be a midkine protein or a protein (specifically binding) to a gene (a molecule comprising more than loo amino acids; eg, an antibody), a peptide (a molecule comprising 1 to 100 amino acids)
  • a gene a molecule comprising more than loo amino acids; eg, an antibody
  • a peptide a molecule comprising 1 to 100 amino acids
  • it may be at least one selected from the group consisting of aptamers, oligonucleotides (eg, siRNAs, shRNAs, microRNAs, aptamers, etc.), small molecule compounds, and pharmaceutically acceptable salts thereof.
  • the antibody comprises at least one contiguous, at least two, at least three, at least four, or at least five (eg, 1 to 30) contiguous to the outside of the primary, secondary or tertiary structure of the midkine protein. , 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10 , 2 to 5, 3 to 30, 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 5, 4 to 30, 4 to 25, 4 to 20 , 4 to 15, 4 to 10, 4 to 5, 5 to 30, 5 to 25, 5 to 20, 5 to 15, 5 to 10, or 5 to 7 amino acid residues)
  • Antibodies in the form of all subtypes of immunoglobulins eg, IgA, IgD, IgE, IgG (IgGl, I G2, IgG3, IgG4), IgM, etc.
  • epitopes antigenic determinants
  • antigen binding fragments thereof ScFv, (scF v) 2
  • the antibody may be a monoclonal antibody or a polyclonal antibody.
  • the antibody may be a mouse antibody, chimeric antibody, humanized antibody or human antibody.
  • the antibody may be derived from a hybridoma or recombinantly or chemically synthesized.
  • the aptamer is 5 to 100 that specifically binds to a specific site of the midkine protein or gene Peptide molecules comprising 5 to 40, 5 to 30, or 5 to 20 amino acids or 5 to 10nt (nucleotides) (e.g.
  • oligonucleotide molecules may be three or more, four or more or five or more contiguous exposed to the primary, secondary or tertiary structure of the midkine gene (eg, 3 to 50, 3 to 45, 3 40 to 40, 3 to 35, 3 to 30, 3 to 25, 3 to 20, 3 to 15 3 to 10, 3 to 5, 4 to 50, 4 to 45 4 to 40 Dog, 4 to 35, 4 to 30, 4 to 25 ⁇ 4 to 20, 4 to 15 4 to 10, 4 to 5, 5 to 50, 5 to 45, 5 to 40 , 5 to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 15, 5 to 10, or 5 to 7 nucleotide sites specifically binding to 3 to 100 , 3 to 90, 3 to 80, 3 to 70, 3 to 60, 3 to 50, 5 to 100
  • the midkine inhibitor is an RNA aptamer (see, for example, Br iti sh Journal of Pharmacology (2014) 896-904), a midkine target anti sense (eg, US 2011). -0159022 Al), anti-midcaine antibodies (e.g. CELLMID limited products (e.g. US 9163081), midkine inhibitory compounds (e.g. AMDon Medchem iMDK (CAS no.
  • Another example is pharmaceuticals for overcoming resistance to radiation therapy and / or enhancing resistance to temozolomide comprising a midkine inhibitor as an active ingredient.
  • Another example is a method of overcoming resistance to temozolomide and / or radiation treatment comprising administering a midkine inhibitor to a subject in need of overcoming resistance or enhancing resistance to radiation therapy and / or temozolomide, and Provide a method of promoting response.
  • the temozolomide and / or resistance to radiation therapy is brain tumor
  • temozolomide Resistance to temozolomide and / or radiation therapy (eg, glioblastoma).
  • the resistance to temozolomide and / or radiation treatment has anti-cancer effects such as reduction of tumor cells, annihilation, reduction of tumor size / volume and / or disappearance during treatment of brain tumors by administration of temozolomide and / or irradiation. It may mean that it does not appear or does not appear therapeutically significant, or does not show anticancer effects depending on the dose and / or dose of temozolomide.
  • Overcoming resistance to the temozolomide and / or radiation treatment is not resistant to the anti-cancer effects such as reduction of tumor cells, annihilation, reduction of tumor size / volume during treatment of brain tumors through administration of temozolomide and / or irradiation It may mean that the tumor is present at a similar or therapeutically significant level as in brain tumors, or that anticancer effects occur depending on the dose and / or radiation dose of temozolomide (ie, proportional to dose and / or dose). have.
  • overcoming resistance to temozolomide and / or radiation treatment may be used in the same sense as enhancing semi-acuity (sensitivity) to temozolomide and / or radiation treatment.
  • Another example provides a pharmaceutical composition for enhancing the efficacy of temozolomide and / or radiation treatment containing a midkine inhibitor as an active ingredient.
  • Another example provides a method of enhancing the efficacy of temozolomide and / or radiation treatment comprising administering a midkine inhibitor to a subject in need of enhancing the efficacy of temozolomide and / or radiation therapy.
  • the improvement of efficacy may be achieved when the anticancer efficacy of brain tumors, such as glioblastoma, is performed by temozolomide administration or radiotherapy alone, respectively.
  • Reduction of tumor cells (glioblastoma) proliferation inhibition and / or increased apoptosis), annihilation, reduction of tumor size / volume and / or abolishment, stem cell capacity inhibition, prevention of recurrence of brain tumors (eg glioblastoma), etc. It may mean any one or more selected from.
  • the subject in need of enhancing the efficacy of the temozolomide and / or radiation treatment may be a brain tumor patient, eg, glioblastoma patient, or a brain tumor patient, such as glioblastoma patient, resistant to temozolomide and / or radiation therapy. have.
  • the radiation treatment refers to radiation treatment applied to conventional brain tumor treatment, and the radiation dose may be various factors such as the patient's age, the patient's health condition, the type of brain tumor, the lesion site, the lesion size, the tumor grade, the disease, and the like. It may be prescribed in consideration of.
  • the radiation treatment may include about lGy to about 6Gy, about lGy to about 4Gy, about lGy to about 2Gy, about 2Gy to about 6Gy, about 2Gy to about 4Gy, or about 4Gy to about 6 Gy and / or one cycle total radiation dose from about 10 Gy to about 60 Gy, about 10 Gy to about 55 Gy, about lOGy to about 50 Gy, about 10 Gy to about 45 Gy, about 10 Gy to about 40 Gy, about lOGy to about 35 Gy, About lOGy to about 30Gy, about lOGy to about 25Gy, or about lOGy to about 20Gy, but is not limited thereto. .
  • the pharmaceutical composition, the combination agent, the first pharmaceutical composition, and / or the second pharmaceutical composition may be used in addition to the active ingredient (midcaine inhibitor and / or temozolomide) such as a pharmaceutically acceptable carrier, diluent, and / or excipient. It may further include an additive.
  • active ingredient midcaine inhibitor and / or temozolomide
  • a pharmaceutically acceptable carrier such as a pharmaceutically acceptable carrier, diluent, and / or excipient. It may further include an additive.
  • the pharmaceutically acceptable carrier which is commonly used in the formulation of drugs, lactose, textose, sucrose, sorbbi, manny, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, fine 1 type selected from the group consisting of crystalline cellulose, polyvinylpyridone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc.
  • It can be more than It may be one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavors, emulsifiers, suspending agents, preservatives and the like commonly used in the preparation of the composition.
  • the combination or pharmaceutical composition may be administered orally or parenterally.
  • parenteral administration it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration and rectal administration.
  • oral administration because proteins or peptides are digested, oral compositions should be formulated to coat the active agent or protect it from degradation in the stomach.
  • the composition may be administered by any device in which the active agent may migrate to the target cell.
  • pharmaceutically effective amount refers to an amount in which a drug can produce a pharmaceutically meaningful effect.
  • the pharmacologically effective amount of the midkine inhibitor and the pharmaceutically effective amount of temozolomide for single administration can be determined by the method of formulation, mode of administration, age, weight, sex, morbidity, food, time of administration, interval of administration, It may be prescribed in various ways depending on factors such as route of administration, rate of excretion and reaction.
  • the pharmaceutically effective amount of the midkine inhibitor and temozolomide for single administration may range from 0.001 to 100 rag / kg, or 0.01 to 10 mg / kg, but is not limited thereto.
  • compositions for single administration may be formulated into one formulation in unit dosage form, may be formulated in appropriate quantities or may be prepared within a multi-dose container.
  • a pharmaceutically effective amount of a midkine inhibitor for one administration and a pharmaceutically effective amount of temozolomide may be included in the packaging containers in basic units, respectively.
  • the interval between the co-administrations is between 6 hours and 30 days, such as between 8 hours and 30 days, 12 hours and 30 days. It may be 24 hours to 30 days, 3 days to 30 days, or 7 days to 14 days, but is not limited thereto.
  • the combination administration sequentially comprises a first pharmaceutical composition comprising a midkine inhibitor as an active ingredient and a second pharmaceutical composition comprising temozolomide as an active ingredient. Concurrently or from 0.1 minute to 60 minutes (eg, from 0.1 minute to 30 minutes, from 0.1 minute to 10 minutes, from 1 minute to 30 minutes, or from 1 to 10 minutes) and the order of administration may be interchanged. Do.
  • the combination or pharmaceutical composition may be formulated in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of axic powders, powders, granules, tablets or capsules, and the like for the formulation of dispersants or It may further comprise a stabilizer.
  • Another example is
  • It provides a method for screening an agent for overcoming the resistance to radiation therapy and / or temozolomide of a brain tumor, or a brain tumor therapy agent resistant to radiation therapy and / or temozolomide.
  • the screening method may further comprise the step of measuring the amount of midkine protein and / or the amount of expression of the midkine gene (mRNA amount) of the brain tumor comparison group cells.
  • the candidate compound is treated with radiotherapy and / or temozole of brain tumor. Selecting as an agent for overcoming resistance to amide or as a candidate for radiation therapy and / or temozolomide resistant brain tumor therapeutic agent.
  • the brain tumor test group cells and brain tumor comparison group cells may be (isolated) brain tumor cells or cultures derived from the same brain tumor patient. remind have.
  • the candidate compound may be selected from the group consisting of various compounds such as proteins, polytide, oligopeptides, polynucleotides, oligonucleotides, various other chemicals, extracts and the like.
  • the amount of the midkine protein or the expression level of the midkine gene can be measured by conventional methods.
  • the amount of the midkine protein can be measured by conventional enzyme reaction, fluorescence, luminescence and / or radiation detection using a compound, antibody, aptamer or the like that binds to the midkine protein, and specifically, immunochromatography Io-unochromatography, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (Floresence immunoassay (FIA), luminescence i ⁇ unoassay (LIA), Western blotting, microarray, etc.), but may be measured by a method selected from the group consisting of, but not limited thereto. no.
  • the expression level of the gene encoding the midkine can be measured by the mRNA level
  • mRNA level can be measured using a conventional genetic analysis method, for example, with the gene Measured using conventional genetic analysis methods, such as polymerizable primers, probes, or aptamers, such as polymerase chain reaction (PCR), f luorescent in si tu hybr idizat ion (FISH), microarray method, and the like. It may be, but is not limited thereto.
  • the primers detect contiguous 5 to 100 bp, such as 10 to 500 bp, 20 to 200 bp, or 50 to 200 bp gene fragments in the sequencing of the gene encoding the midkine (full length DNA, cDNA, or mRNA). It may be possible to hybridize (eg, complementary to) the contiguous 5-100 bp, e.g., 5-50 bp, 5-30 bp, or 10-25 bp region of each of the 3'-end and 5 1 -end of the gene fragment. ) May be a primer pair including a nucleotide sequence.
  • the probe or aptamer has a total length of 5 to 100 bp
  • Binding or hybridizing e.g., complementary
  • a contiguous sequence of 5 to 100 bp, 5 to 50 bp, 5 to 30 bp, or 5 to 25 bp gene fragment in the nucleotide sequence of the gene encoding (full length DNA, cDNA, or mRNA) ) May have a nucleotide sequence.
  • the term 'combinable' may mean that the gene region may be bound by chemical and / or physical bonds such as covalent bonds, and the term “saturable” may refer to a nucleotide sequence of the gene region. As described above, for example, by 90% or more, 95% or more, 98% or more, 99% or more, or 100% by sequence complementarity, it may mean that complementary binding is possible.
  • ⁇ Effects of the Invention e.g., complementary to a contiguous sequence of 5 to 100 bp, 5 to 50
  • midcaine a self-secreting growth factor of glioblastoma cells, on the proliferation of tumor cells, we suggest the utility as a target for new glioblastoma treatment, thereby providing a new use of midcaine as a target for new drug development. do.
  • the use of midkine inhibitors in the treatment of brain tumors, such as glioblastoma, alone or in combination with temozolomide and / or radiation therapy can achieve an elevated anticancer effect, and treatment of conventional brain tumors such as temozolomide and / or radiation therapy. Treatment or life extension of brain tumors (eg, glioblastoma) that are resistant to is possible. [Brief Description of Drawings]
  • 1 is an image showing the results of staining the midkine protein by immunohistochemical staining (I ⁇ unohi stochemi stry) in tumor tissue and normal tissue of glioblastoma patients (bar: 100 ⁇ ).
  • FIG. 2 is an image showing the results of observing midkine protein expression levels in tumor cells of glioblastoma patients with West blot.
  • Figure 3 is a graph showing the relative absorbance of N827 glioblastoma cell cultures in the midkine (MDK) neutralizing antibody and IgG (control) treatment according to the antibody treatment concentration.
  • Figure 4 is the MDK neutralization in three glioblastoma cell cultures N131, 047T, and N827 It is a graph.
  • Figure 5 shows the results of microscopic observation of the cell appearance on the 11th day after treatment with MDK neutralizing antibody and IgG (control) to N131, 047T, and N827 three glioblastoma cells (photo; right) and the number of tumor cells Graph (left).
  • Figure 6 shows the Western blot results upon shRNA treatment for MDK in N131 and N827 two glioblastoma cells.
  • FIG. 7 is a graph showing the cell proliferation rate (expressed as Relative ATP) when N827 glioblastoma cells were treated with shRNA against MDK compared with that without treatment with shRNA (NT).
  • FIG. 8 shows the tumor cell formation (top), the graph showing the number of cells without tumor cell formation (middle), and the result of quantification thereof (bottom) upon shRNA treatment for MDK in N131 glioblastoma cells.
  • FIG. 9 shows tumor cell formation (top), graph showing the number of cells without tumor cell formation (middle), and numerical results (bottom) upon shRNA treatment for MDK in N827 glioblastoma cells.
  • 10A to 10C show cell proliferation result graphs and microscopic observation images when the cells of N131, 047T, and N827 glioblastoma cells were treated with MDK-targeting neutralizing antibody and temozolomide (TMZ) alone. Not very good.
  • FIG. 11 is a photograph showing the cell-formation of the glioblastomas by the irradiation dose (control) and the combination of the irradiation and the midkine target antibody (ant i-MDK).
  • FIG. 12 is a graph showing the cell number of each glioblastoma by irradiation dose (control) and the combination of irradiation and midkine target antibody (ant i-MDK).
  • FIG. 13 shows midkine knockdown (shMDK infection) N827 cells and N827 cells
  • Example 1 Confirmation of midkine (MDK) expression in glioblastoma patient tissue
  • glioblastoma patients The level of midkine protein expression in cancerous and normal tissues of glioblastoma patients was confirmed by immunohistochemistry (I ⁇ unohistochemistry). Cancer tissue obtained from glioblastoma patients (named NS234 Tumor & NS320 Tumor; provided by Samsung Seoul Hospital) and normal tissues (named NS234 Normal & NS320 Normal; provided by Samsung Seoul Hospital) were fixed in paraffin and treated with deylene for 15 minutes of xylene. After the f inizat ion, the mixture was hydrated by treating with 100% (v / v) ethanol for 10 minutes, 95% (v / v) ethanol for 10 minutes, and dH20 for 5 minutes. Antigen masking was performed by boiling the hydrated tissue for 10 minutes.
  • FIG. 1 shows MDK expression levels in normal brain tissues and brain tumor tissues of the same patient through immunochemical staining, and it can be seen that MDK levels are specifically increased in brain tumor tissues.
  • the Western blotting was performed by loading each cell lysate in an amount of 30 ug on a 12% SDS page gel and gel running 100V), and then blocking 1 hour at room temperature with 53 ⁇ 4) (v / v) BSA after 1 hour of transfer (100V).
  • Midkine (Abeam, ab52637) and GAPDH cell signaling, 2118) were treated with primary antibody for 2 hours at room temperature, washed three times for 10 minutes with 1XTBST, and then anti-rabbit HRP linked secondary antibody (cell signaling, 7074S) was stored at room temperature. 1 hour of treatment. Subsequently, it was developed by spraying a solution of Super Signal West Dura Extended Duration Substrate CTermo scientific (34075) on a membrane washed three times for 10 minutes with 1XTBST.
  • Example 2 Effect of Midcaine Neutralizing Antibody Treatment on Glioblastoma Tumor Cells In order to determine the concentration of midcaine neutralizing antibody, the above Example
  • ELISA was performed using the culture medium of N827 glioblastoma cells prepared in step 2. ELISA was conducted using the Human Midkine Standard ELISA Development Kit (PeproTech, 900-K190; reagents used in the following tests and antibodies included in the kit).
  • the capture antibody When preparing an ELISA plate, the capture antibody was diluted to 1 / ml in PBS, dispensed with 100 ⁇ per lwell, sealed, and incubated overnight at room temperature. After incubation, Suction was carried out, and 300 ⁇ wash buffer (0.05% (v / v) Tween-20 in PBS) was added per lwell and washed four times. After the final washing was finished, placed on a paper towel and dried to remove the buffer completely, 300 blocks of buffer buffer (1% ( ⁇ / ⁇ ) BSA in PBS) was added to each well and incubated at room temperature for 1 hour. Then, after the suction again, washed four times with 300 ⁇ wash buffer per lwell.
  • 300 ⁇ wash buffer 0.05% (v / v) Tween-20 in PBS
  • the experimental group is to calculate the absorbance of pure cells themselves, except for Standard (2ng, lng, 0.5ng, 0.25ng, 0.125ng, Ong) and media only ), IgG (Normal Rabbit IgG (cat. AB-105-C, R & D Systems)) treatment group (control; 0, 0.156, 0.625, 2.5, 10zg / ml), and Midkine target neutralizing antibody (MK (H-65) Antibody (cat.sc-20715, SANTA CRUZ BIOTECHNOLOGY) treatment group (0, 0.156, 0.625, 2.5, 10 g / ml), each group of samples on a 96well plate with 100 ⁇ per lwell (for each sample) 3 wells prepared) was dispensed and incubated at room temperature for 2 hours. After completion of the incubation, the sample was suctioned and washed four times with 300 id wash buffer per lwell.
  • Standard 2ng, lng, 0.5ng, 0.25ng, 0.
  • the detection antibody was diluted with 1 g / ml and dispensed at 100 ⁇ / lwell and incubated at room temperature for 2 hours. After 2 hours, suction and washing were performed 4 times with 300 ⁇ wash buffer per lwell. 5.5 After diluting Avidin-HRP 1: 2000 in 11 ml dilution, 100 ⁇ for each well. After the sample was suctioned and washed four times with 300 f wash buffer per lwell, 100 wells of substrate solution was dispensed into each well to confirm color development (ELISA plate reader at 405 nm with wavelength correction set at ' 650 nm).
  • N131 (labeled 'N131' in FIG. 4, 047T (labeled '047' in FIG. 4)) and N827 (FIG.
  • glioblastoma cells (labeled as 'N827') were seeded by 3 wells for each cell line, each containing 5000 cells in lOOul cell culture per lwell, and IgG (10 // g / ml) and Midkine target neutralizing antibodies ( MKCH—65) Antibody (cat.sc-20715, SANTA CRUZ ⁇ (: ⁇ ⁇ ⁇ / / ⁇ , respectively) were treated.
  • EZ-Cytox (EZ-cytox Cell Viability Assay Kit; Note) Daeil Biotech) was treated in an amount of 10ul per lwell, incubated at 37 ° C for 1 hour, and the degree of cell proliferation was measured by measuring absorbance at 450nm.
  • N131 Neurogenital-A Medium (cat. 10888-022, gibco) + N-2 Supplement (100X) (cat. 17502-048, life technologies) + B-27 Supplement (50X) (cat. 17504-044, life technologies) + human FGF-basic (cat.AF-100-18B, Peprotech) + human EGF (cat.AF-100-15, Peprotech) + Penicillin-Streptomycin (cat. 15140-163, gibco) + L-glut amine 200mM (cat.
  • FIG. 5 The results are shown in FIG. 5.
  • the right photograph is a photomicrograph of the formed tumor cells (200 ⁇ )
  • the left graph is a graph showing the number of tumor cells observed per well.
  • the midkine gene was knocked down to test how the reduction in the gene level of midcaine affects glioblastoma cells. Specifically, after infecting Midkine knockdown virus to N131 and N827 cells (infected with 40ul of virus in 5X10 6 cells), the degree of protein expression was confirmed by Western blot.
  • the Midkine knockdown virus contains two kinds of shRNAs, respectively:
  • shRNA 210 (denoted shRNA1): ShRNA252 (denoted shRNA2):
  • the Midkine knockdown virus was purchased from Sigma-Aldrich in the form of shRNA Glycerol stock (shRNA 210 (denoted shRNA1; TRCN0000331210) and ShRNA252 (denoted shRNA2; TRCN0000331252)).
  • the obtained midkine gene was knocked down on N827 glioblastoma cells and N827 cells not knocked down (NT) on a 384well plate.
  • the measured cell number results were statistically analyzed using the SPSS statistical program (Statistical Package for the Social Sciences; IBM) to determine how many cells should exist in each group to form tumor cells.
  • SPSS statistical program Statistical Package for the Social Sciences; IBM
  • FIG. 8 shows the results obtained for the N131 cell line (top: micrograph (200 ⁇ ); middle: graph showing the number of cells without tumor cells; bottom: statistical analysis results).
  • top micrograph (200 ⁇ ); middle: graph showing the number of cells without tumor cells; bottom: statistical analysis results.
  • tumor cell formation ability after treatment of MDK gene target inhibitor (shRNA for MDK) is significantly reduced compared to the comparison group (shRNA untreated group for MDK: NT).
  • 'Estimate' in the lower table means the average number of cells that can form tumor cells.
  • shRNA-untreated group (NT) for MDK only 61.7 cells can form tumor cells, while shRNA-treated group (MDK Because cells must be present to form tumor cells, tumor cell formation ability when treated with shRNA against MDK (ie, when inhibition of MDK expression) is significantly reduced compared to the non-treated group (NT). Can be.
  • Figure 9 shows the results obtained for the N827 cell line (top: micrograph (200X); middle: graph showing the number of cells without tumor cells; bottom: statistical analysis results).
  • top micrograph (200X); middle: graph showing the number of cells without tumor cells; bottom: statistical analysis results.
  • NT comparison group
  • Prol i ferat ion assay was performed. Three glioblastoma cells of N131, 047, and N827 were placed on a 96wel l plate per lwell lOOul cell culture solution (Neurobasal-A Medium (cat 10888-022, gibco) + N-2 Supp 1 ement (50X) (cat .17504-044, life technologies) + human FGF-basic (cat.AF-100-18B, Peprotech) + human EGF (cat .AF-100-15, Peprotech) + Penici 11 in -Streptomycin (cat.
  • the test group included only culture medium (control: in this case, IgG was treated with a control antibody at a concentration of 5 ug / ml) and temozolomide ⁇ 10201011 ⁇ (16 (0; 12577, Sigma-Aldr ich) alone treatment) Group (treatment concentration: 0, 1, 3.9, 15.6, 62.5, 250, lOOOOmol; in this case, IgG was treated with a concentration of 5 ug / ml as a control antibody), midkine target antibody (MK (H-65) Antibody ( cat.sc-20715, SANTA CRUZ BIOTECHNOLOGY)) alone treatment group (treatment concentration: 5ug / ml), and temozolomide and midcaine target antibody treatment group (temozolomide treatment concentration: 0, 1, 3.9, 15.6, 62.5 , 250,
  • the antibody was treated at lOug / ml (see Example 2).
  • the midkine target antibody was co-treated with temozolomide, the proliferation of glioblastoma cells was reduced.
  • the antibody throughput was adjusted to 5 ug / ml to take into account the effect on.
  • EZ-Cytox EZ-cytox Cell Viability Assay Kit; ⁇ Daily Biotech
  • FIGS. 10A (N131), 10B (047), and 10C (N827) show the absorbance ratios (relative values) of each experimental group relative to the absorbance ('1') of the control (treated with control IgG only).
  • 10A to 10C show graphs showing the degree of cell proliferation for each cell line and micrographs of the cells.
  • the cancer cells were dose-dependently depending on the concentration of temozolomide compared with the administration of the midkine target neutralizing antibody and temozolomide (TMZ) and administration of temozolomide alone (indicated by control IgG). It can be seen that the killing ability is increased (see the graph provided for each cell line).
  • the cells were stabilized by incubating N827 glioblastoma cells at 5 ⁇ 10 5 cel l per wel l in 6weil cel l culture p 1 at e (cat. 140675, Termo scient if ic) and incubating for 24 hours. After 24 hours, the prepared cells were transferred to IBL 437C blood irradiator CCIS US, Inc. , Bedford, MA) to obtain OGy, 2Gy, 4Gy, 6Gy radiation, respectively. Midkine target antibody (cat. Sc-20715, SANTA CRUZ BIOTECHNOLOGY) was treated at a concentration of 5 ng / ml.
  • Figure 11 is a microscopic observation of the tumor cell formation obtained in the test, the degree of the formation of the cell sphere of glioblastoma cells can be said to show the stem cell capacity. As shown in the upper part of FIG. 11 (control; only radiation treatment), radiation treatment alone did not reduce stem cell function of glioblastoma cells, but the radiation treatment and midkine target antibody administration were performed. In parallel (ant i-MDK), it can be seen that the stem cell capacity of glioblastoma cells is significantly reduced.
  • FIG. 12 is a graph showing the number of obtained cell cells. As shown in FIG. 12, only glioblastoma cells were treated with radiotherapy (control), but no cell death (reduction) effect was observed regardless of the radiation dose (ie, resistance to radiation treatment). When the antibody was administered in parallel (Ant i-MDK), it can be seen that a significant cancer cell death (reduction) effect is shown, the cancer cell death is increased in proportion to the radiation dose. In particular, even when the radiation dose was 0 and only administration of the midkine target antibody showed about 40% reduction of cancer cells, the anticancer effect on the cancer cells resistant to the radiation treatment by the midkine target treatment (tumor formation inhibitory effect). It was confirmed that can be obtained).
  • Example 6 Confirmation of In Vivo Tumor Suppression Capability by Midkine Gene Knockdown
  • a cerebral stereotactic mouse model was prepared to confirm the decrease of tumor formation ability when the midkine gene was knocked down.
  • a BALB / c-nude mouse (Oriental Bio, female) was used with a brain stereotactic injector (coordinate AP 0.5 mm, ML + 1.7 ⁇ s, DV-3.2 ⁇ s) to express N827 cells (hereinafter referred to as N827) 1 ⁇ 10 4 cels per subject.
  • the amount of l was injected for 10 minutes using a Hami lton syringe. At this time, as the N827 cells N827 cells were used.
  • mice were injected with N827 cells infected with a non target vector (cat. SHC016, Sigma-Aldrich) virus.
  • a total of 20 animals were prepared, with 10 animals in each group.
  • the prepared brain stereotactic mouse model was measured once daily weight, and when the weight of the control group is reduced by more than 20%, the brain by sacrificing two heads each of the experimental group (shM infection N827 cell injection) brain stereotype mouse model and control mouse model Extracted, fixed with 4% Paraformaldehyde Solution (cat.HP2031, Bio World), and confirmed the size of the tumor by H & E (Hematoxylin and Eosin) staining.
  • H & E Hematoxylin and Eosin
  • Hematoxylin and Eosin (H & E) staining methods are as follows:
  • the remaining cerebral stereotactic mouse models were sacrificed continuously at 20% or more weight loss, and brains were extracted and stored in 4% Paraformaldehyde Solution (cat.HP2031, Bio World Co., Ltd.) for later experiments.
  • FIG. 13 As a result of confirming the tumor size through the H & E (Hematoxylin and Eosin) staining (two heads for each group) are shown in FIG. 13 (bar: 2 ms). As shown in FIG. 13, as a result of confirming the size of the tumor throughout the year, the size of the tumor formed in the brain was significantly reduced in the shMDK-treated group (shMDK) compared to the control group (NT) (ie, tumor formation ability was suppressed).
  • shMDK shMDK-treated group
  • NT control group
  • the survival period of each mouse model (the period until the weight of the last surviving mouse is reduced by 20%) is measured and shown in FIG. 14. As shown in FIG. I can see that.

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Abstract

L'invention concerne: une composition pharmaceutique pour le traitement et/ou la prévention de tumeurs cérébrales, contenant, en tant que principe actif, un inhibiteur de midkine ou une combinaison d'un inhibiteur de midkine et de témozolomide; et l'utilisation d'un inhibiteur de midkine pour combattre la résistance au traitement par le témozolomide et/ou à la radiothérapie.
PCT/KR2016/009659 2016-07-19 2016-08-30 Composition pharmaceutique pour le traitement ou la prévention de tumeurs cérébrales, contenant un inhibiteur de midkine Ceased WO2018016674A1 (fr)

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