WO2015194643A1 - Inhibiteur de la croissance cellulaire dépendant de pdgf, procédé d'inhibition de la croissance cellulaire dépendant de pdgf, inhibiteur de la dispersion des cellules, procédé d'inhibition de la dispersion des cellules, renforçateur de l'activité du témozolomide, et agent antitumoral - Google Patents

Inhibiteur de la croissance cellulaire dépendant de pdgf, procédé d'inhibition de la croissance cellulaire dépendant de pdgf, inhibiteur de la dispersion des cellules, procédé d'inhibition de la dispersion des cellules, renforçateur de l'activité du témozolomide, et agent antitumoral Download PDF

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WO2015194643A1
WO2015194643A1 PCT/JP2015/067651 JP2015067651W WO2015194643A1 WO 2015194643 A1 WO2015194643 A1 WO 2015194643A1 JP 2015067651 W JP2015067651 W JP 2015067651W WO 2015194643 A1 WO2015194643 A1 WO 2015194643A1
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cell
amino acid
thr
pdgf
peptide
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PCT/JP2015/067651
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Japanese (ja)
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文雄 深井
哲哉 山本
浩明 兒玉
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学校法人東京理科大学
国立大学法人筑波大学
国立大学法人佐賀大学
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Priority to JP2016529523A priority Critical patent/JPWO2015194643A1/ja
Publication of WO2015194643A1 publication Critical patent/WO2015194643A1/fr

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    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]

Definitions

  • the present invention relates to a PDGF-dependent cell growth inhibitor, a PDGF-dependent cell growth inhibitory method, a cell dispersion inhibitor, a cell dispersion inhibitory method, a temozolomide activity enhancer, and an antitumor agent.
  • Glioma a brain tumor derived from glial cells (glial cells), is extremely malignant and has a cure rate of less than 10%.
  • the reason is that the high proliferation ability of glioma cells and the active migration / invasion ability make surgical removal of the tumor difficult. Therefore, if the growth, migration, and infiltration of tumor cells that make surgical removal of the tumor difficult can be effectively suppressed, surgical treatment becomes possible, and an improvement in the cure rate can be expected. Moreover, if the effect of the existing drug can be effectively enhanced, effects such as improvement of the cure rate and reduction of side effects can be expected.
  • Patent Documents 1 and 2 As a means for suppressing tumor growth and invasion, it has been reported that peptides comprising an amino acid sequence derived from an extracellular matrix, fibronectin, and having cell adhesion inhibitory activity are useful as cancer metastasis inhibitors (for example, Patent Documents 1 and 2). In addition, it has been reported that a peptide comprising an amino acid sequence derived from fibronectin is useful as an activity enhancer of an existing anticancer agent (for example, Patent Document 3).
  • Patent Documents 1 and 2 do not demonstrate the effect of Examples as cancer metastasis inhibitors of peptides having cell adhesion inhibitory activity.
  • Patent Document 3 does not discuss the combined use of a peptide comprising an amino acid sequence derived from fibronectin and temozolomide used as a therapeutic agent for glioma.
  • the present inventors are useful for inhibiting the growth or dispersion of tumor cells, PDGF-dependent cell growth inhibitors, methods for inhibiting PDGF-dependent cell proliferation, cell dispersion inhibitors, methods for inhibiting cell dispersion,
  • An object is to provide a temozolomide activity enhancer and an antitumor agent.
  • a PDGF-dependent cell growth inhibitor comprising the following peptide (a) or (b) as an active ingredient.
  • (A) Thr-Glu-Ala-Thr-Ile-Thr-Gly-Leu-Glu-Pro-Gly-Thr-Glu-Tyr-Thr-Ile-Tyr-Val-Ile-Ala-Leu SEQ ID NO: 1
  • a peptide comprising the amino acid sequence represented A peptide comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted or added in the amino acid sequence (a) and having a PDGF-dependent cell growth inhibitory action.
  • ⁇ 2> The PDGF-dependent cell growth inhibitor according to ⁇ 1>, wherein the cell is at least one cell selected from the group consisting of sarcoma and carcinoma that expresses a PDGF receptor.
  • ⁇ 3> The PDGF-dependent cell growth suppression according to ⁇ 1> or ⁇ 2>, wherein the cell is at least one cell selected from the group consisting of glioma, fibrosarcoma, osteosarcoma, breast cancer and lung cancer. Agent.
  • ⁇ 4> A method for inhibiting PDGF-dependent cell growth, which comprises contacting the cell with the PDGF-dependent cell growth inhibitor according to any one of ⁇ 1> to ⁇ 3>.
  • a cell dispersion inhibitor comprising the following peptide (a) or (b) as an active ingredient.
  • A Thr-Glu-Ala-Thr-Ile-Thr-Gly-Leu-Glu-Pro-Gly-Thr-Glu-Tyr-Thr-Ile-Tyr-Val-Ile-Ala-Leu (SEQ ID NO: 1)
  • a peptide comprising the amino acid sequence represented.
  • B A peptide comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted or added in the amino acid sequence (a) and having a cell dispersion inhibitory action.
  • ⁇ 6> The cell dispersion according to ⁇ 5>, wherein the cell is at least one cell selected from the group consisting of a sarcoma and a carcinoma that causes dissociation of a cell from a population formed by cell-cell adhesion or an aggregation. Inhibitor.
  • ⁇ 7> The cell dispersion inhibitor according to ⁇ 5> or ⁇ 6>, wherein the cell is at least one cell selected from the group consisting of glioma, fibrosarcoma, osteosarcoma, breast cancer and lung cancer.
  • ⁇ 8> A method for inhibiting cell dispersion, comprising contacting the cell dispersion inhibitor according to any one of ⁇ 5> to ⁇ 7> with cells.
  • a temozolomide activity enhancer comprising the following peptide (a) or (b) as an active ingredient.
  • (A) Thr-Glu-Ala-Thr-Ile-Thr-Gly-Leu-Glu-Pro-Gly-Thr-Glu-Tyr-Thr-Ile-Tyr-Val-Ile-Ala-Leu SEQ ID NO: 1
  • a peptide comprising the amino acid sequence represented.
  • (B) A peptide having an activity of enhancing temozolomide activity, comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted or added in the amino acid sequence (a).
  • An antitumor agent comprising temozolomide and the following peptide (a) or (b) as active ingredients.
  • a PDGF-dependent cell growth inhibitor a PDGF-dependent cell growth inhibitory method, a cell dispersion inhibitor, a cell dispersion inhibitory method, and a temozolomide activity enhancer useful for inhibiting tumor cell growth or dispersion And an anti-tumor agent is provided.
  • a numerical range indicated by using “to” indicates a range including the numerical values described before and after “to” as the minimum value and the maximum value, respectively.
  • the left side is the N-terminal side
  • the amino acid residue is represented by one letter (for example, “G” for glycine residue) or three letter (for example, “Gly” for glycine residue) well known in the art. ) In some cases.
  • the PDGF-dependent cell growth inhibitor of the present invention contains the following peptide (a) or (b) (hereinafter also referred to as a specific peptide) as an active ingredient.
  • (A) Thr-Glu-Ala-Thr-Ile-Thr-Gly-Leu-Glu-Pro-Gly-Thr-Glu-Tyr-Thr-Ile-Tyr-Val-Ile-Ala-Leu (SEQ ID NO: 1) A peptide comprising the amino acid sequence represented.
  • B A peptide comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted or added in the amino acid sequence (a) and having a PDGF-dependent cell growth inhibitory action.
  • the specific peptide is (b): a peptide in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a), the number of amino acid residues to be deleted, substituted or added is the specific peptide Is not particularly limited as long as it has a PDGF-dependent cell growth inhibitory effect.
  • the number is 1 to 9, preferably 1 to 6, and more preferably 1 to 4.
  • the total number of amino acid residues of the peptide (b) is preferably 30 or less, more preferably 28 or less, and 25 or less. More preferred.
  • the amino acid sequence of the peptide (a) above is derived from the fibronectin type III domain constituting fibronectin, which is one of the extracellular matrix protein molecules.
  • the present inventors have found that a peptide having the above amino acid sequence has an action of suppressing PDGF-dependent cell growth.
  • PDGF-dependent means that cell proliferation is accompanied by expression of platelet-derived growth factor (PDGF) receptor. Whether or not PDGF is expressed can be confirmed by Western blotting, flow cytometry or the like using an anti-PDGF receptor antibody.
  • the PDGF-dependent cell growth inhibitor of the present invention has an action of suppressing PDGF-dependent cell growth. That is, in tumors where PDGF receptor expression is observed, the expression of tenascin-C, which is an extracellular matrix having the property of being temporarily expressed during inflammation or lesion, is significantly increased.
  • a peptide released from tenascin-C (TNIIIA2) has an action of activating integrin, which is a cell adhesion molecule, and this enhances PDGF-dependent growth of tumor cells.
  • the specific peptide contained in the PDGF-dependent cell growth inhibitor of the present invention has an effect of inactivating integrin.
  • TNIIIA2 As a result, it is considered that the integrin activation action of TNIIIA2 is suppressed and the growth of tumor cells is suppressed.
  • TNIIIA2 see J.A. Biol. Chem. , Vol. 282, pp. No. 34929-34937, (2007), and the like.
  • tumors with PDGF-dependent cell proliferation include gliomas, sarcomas such as fibrosarcomas and osteosarcomas, and cancer types such as breast cancer and lung cancer.
  • Ciliary cell astrocytoma, diffuse astrocytoma, anaplastic astrocytoma and glioblastoma, oligodendroglioma, ependymoma, and choroid plexus classified as astrocytic tumors as glioma Papilloma may be mentioned.
  • the degree of PDGF expression is not particularly limited as long as the effect of the present invention can be obtained, but the effect of the present invention is more remarkable in tumors that express PDGF more strongly.
  • glioblastoma having high malignancy among gliomas is known to express PDGF at a high level, and the effect of the present invention is particularly remarkable.
  • the specific peptide contained in the PDGF-dependent cell growth inhibitor of the present invention may be variously modified depending on the application.
  • a water-soluble polymer such as polyethylene glycol, a sulfate group, a water-soluble amino acid, or the like may be linked to the terminal of a specific peptide to improve water solubility or multimerize.
  • each of the amino acid residues constituting the specific peptide may be either L-form or D-form as long as the effect of the present invention is achieved. From the viewpoint of suppressing the degradation of the specific peptide in the body, it is preferable that at least a part of the amino acid sequence (for example, Glu at the second position) is D-form.
  • the method for producing the specific peptide is not particularly limited, and may be either a genetic engineering method or an organic synthetic chemical method.
  • the PDGF-dependent cell growth inhibitor of the present invention may contain components other than the specific peptide depending on the use mode.
  • the components other than the specific peptide include media generally used for the preparation of drugs and additives for pharmaceutical preparations.
  • the type of the medium and the additive for formulation is not particularly limited.
  • the medium include a solid medium (for example, gelatin and lactose) and a liquid medium (for example, alcohol, water, and physiological saline).
  • the additive for preparation include excipients, disintegrants, binders, lubricants, surfactants, buffers, solubilizers, stabilizers, tonicity agents, and the like.
  • the form of the PDGF-dependent cell growth inhibitor of the present invention is not particularly limited, and can be selected according to use.
  • forms suitable for oral administration such as tablets, granules, powders, capsules, suspensions, syrups, emulsions, limonades, ampoules for injection, freeze-dried powder for injection, dry powder for pulmonary administration Etc.
  • the method for inhibiting PDGF-dependent cell proliferation of the present invention comprises contacting cells with the PDGF-dependent cell proliferation inhibitor of the present invention.
  • Contacting a cell means bringing a cell exhibiting PDGF-dependent proliferation into contact with a specific peptide contained in the PDGF-dependent cell growth inhibitor of the present invention. Contact with a tumor or cancer tissue composed of cells exhibiting proliferation.
  • the method for bringing the PDGF-dependent cell growth inhibitor into contact with the cells is not particularly limited, and examples thereof include surgical treatment such as oral administration, intravenous administration, and indwelling.
  • the amount of the PDGF-dependent cell growth inhibitor to be brought into contact with the cell is not particularly limited, and can be selected according to the state of the cell, the type and amount of other components used with the specific peptide, and the like.
  • the cell dispersion inhibitor of the present invention contains the following peptide (specific peptide) (a) or (b) as an active ingredient.
  • the specific peptide is (b): a peptide in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a), the number of amino acid residues to be deleted, substituted or added is the specific peptide If it is a range which has a cell dispersion
  • the number is 1 to 9, preferably 1 to 6, and more preferably 1 to 4.
  • the total number of amino acid residues of the peptide (b) is preferably 30 or less, more preferably 28 or less, and 25 or less. More preferred.
  • the present inventors have found that the specific peptide having the amino acid sequence has an action of suppressing cell dispersion.
  • the dispersion of cells By suppressing the dispersion of cells forming the tumor, the migration and invasion of the tumor is effectively suppressed. As a result, it is possible to apply a technique such as surgical extraction, and the range of choice of treatment method is expanded.
  • “Dispersion” of cells means that individual cells dissociate from a group formed by cell-cell adhesion between cells or from a gathered state. The malignant tumor cells dissociate from the primary tumor tissue by being “dispersed” and infiltrate and metastasize to surrounding tissues.
  • cell dispersion inhibitor of the present invention has an action of suppressing cell dispersion is not clear, but is presumed as follows. That is, cell dispersion is induced by the integrin activation action of a peptide (TNIIIA2) released from tenascin C expressed at the tumor site. Therefore, it is considered that cell dispersion is suppressed by applying the cell dispersion inhibitor of the present invention containing a specific peptide having an action of inactivating integrin.
  • tumors in which cell dispersion is observed include sarcomas such as glioma, fibrosarcoma and osteosarcoma, and cancer types such as breast cancer and lung cancer.
  • Ciliary cell astrocytoma, diffuse astrocytoma, anaplastic astrocytoma and glioblastoma, oligodendroglioma, ependymoma, and choroid plexus classified as astrocytic tumors as glioma Papilloma may be mentioned.
  • the degree of cell dispersion is not particularly limited as long as the effect of the present invention is obtained, but the effect of the present invention is more remarkable in a tumor in which cell dispersion is more active.
  • glioblastoma having high malignancy among gliomas is known to have active cell dispersion, and the effect of the present invention is particularly remarkable.
  • the specific peptide contained in the cell dispersion inhibitor of the present invention may be variously modified depending on the application.
  • a water-soluble polymer such as polyethylene glycol, a sulfate group, a water-soluble amino acid, or the like may be linked to the terminal of a specific peptide to improve water solubility or multimerize.
  • each of the amino acid residues constituting the specific peptide may be either L-form or D-form as long as the effect of the present invention is achieved. From the viewpoint of suppressing the degradation of the specific peptide in the body, it is preferable that at least a part of the amino acid sequence (for example, Glu at the second position) is D-form.
  • the method for producing the specific peptide is not particularly limited, and may be either a genetic engineering method or an organic synthetic chemical method.
  • the cell dispersion inhibitor of the present invention may contain components other than the specific peptide depending on the use mode.
  • the components other than the specific peptide include media generally used for the preparation of drugs and additives for pharmaceutical preparations.
  • the type of the medium and the additive for formulation is not particularly limited.
  • the medium include a solid medium (for example, gelatin and lactose) and a liquid medium (for example, alcohol, water, and physiological saline).
  • the additive for preparation include excipients, disintegrants, binders, lubricants, surfactants, buffers, solubilizers, stabilizers, tonicity agents, and the like.
  • the form of the cell dispersion inhibitor of the present invention is not particularly limited and can be selected depending on the application.
  • forms suitable for oral administration such as tablets, granules, powders, capsules, suspensions, syrups, emulsions, limonades, ampoules for injection, freeze-dried powder for injection, dry powder for pulmonary administration Etc.
  • the method for inhibiting cell dispersion of the present invention includes contacting the cell dispersion inhibitor of the present invention with cells.
  • Contacting a cell means a cell forming a population or a cluster by cell-cell adhesion, or a cell dissociated from a population or a cluster, and a specific peptide contained in the cell dispersion inhibitor of the present invention In a tumor or cancer tissue in which dissociation of cells from a population or collection has not yet been observed but is highly likely to occur or dissociation has already been observed. Including contacting.
  • the method for bringing the cell dispersion suppression into contact with the cells is not particularly limited, and examples thereof include surgical treatment such as oral administration, intravenous administration, and indwelling.
  • the amount of the cell dispersion inhibitor to be brought into contact with the cell is not particularly limited, and can be selected according to the state of the cell, the type and amount of other components used together with the specific peptide.
  • the temozolomide activity enhancer of the present invention contains the following peptide (specific peptide) (a) or (b) as an active ingredient.
  • B A peptide comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted or added in the amino acid sequence (a), and having a temozolomide activity enhancing action.
  • the specific peptide is (b): a peptide in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a),
  • the number of amino acid residues to be deleted, substituted or added is the specific peptide Is not particularly limited as long as it has a temozolomide activity enhancing action.
  • the number is 1 to 9, preferably 1 to 6, and more preferably 1 to 4.
  • the total number of amino acid residues of the peptide (b) is preferably 30 or less, more preferably 28 or less, and 25 or less. More preferred.
  • the temozolomide activity enhancer of the present invention is an antitumor agent of temozolomide (3,4-dihydro-3-methyl-4-oxoimidazo [5,1-d] [1,2,3,5] tetrazine-8-carboxamide). Strengthen the action. Temozolomide is an oral drug belonging to an alkylating agent, and has a low molecular weight and can pass through the blood-brain barrier and is used as a therapeutic agent for brain tumors.
  • the present inventors have found that a specific peptide having the above amino acid sequence enhances the antitumor action of temozolomide. That is, when the temozolomide activity enhancer of the present invention containing a specific peptide as an active ingredient is used in combination with temozolomide, it is possible to obtain an antitumor effect that exceeds the case where temozolomide or the specific peptide is used alone. Moreover, by using the temozolomide activity enhancer of the present invention in combination with temozolomide, the amount of temozolomide used can be reduced while achieving a sufficient antitumor effect, and side effects caused by temozolomide can be reduced.
  • the method for administering the temozolomide activity enhancer of the present invention is not particularly limited, and can be selected according to use.
  • surgical treatment such as oral administration, intravenous administration, and indwelling can be exemplified.
  • temozolomide is generally administered orally
  • temozolomide activity enhancers are also preferably administered orally.
  • the temozolomide activity enhancer and temozolomide may be administered simultaneously or at intervals.
  • the temozolomide activity enhancer of the present invention may contain components other than the specific peptide according to the use mode, and the form thereof is not particularly limited and can be selected according to the use. Specific examples of components and forms that may be contained in addition to the specific peptide are the same as the specific examples described for the PDGF-dependent cell growth inhibitor or cell dispersion inhibitor of the present invention.
  • the antitumor agent of the present invention contains temozolomide and the following peptide (specific peptide) (a) or (b) as active ingredients.
  • a) Thr-Glu-Ala-Thr-Ile-Thr-Gly-Leu-Glu-Pro-Gly-Thr-Glu-Tyr-Thr-Ile-Tyr-Val-Ile-Ala-Leu SEQ ID NO: 1
  • B A peptide comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted or added in the amino acid sequence (a), and having a temozolomide activity enhancing action.
  • the specific peptide is (b): a peptide in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a),
  • the number of amino acid residues to be deleted, substituted or added is the specific peptide Is not particularly limited as long as it has a temozolomide activity enhancing action.
  • the number is 1 to 9, preferably 1 to 6, and more preferably 1 to 4.
  • the total number of amino acid residues of the peptide (b) is preferably 30 or less, more preferably 28 or less, and 25 or less. More preferred.
  • the antitumor agent of the present invention may be a mixed agent containing temozolomide and a specific peptide as active ingredients, or a combination of drugs each containing temozolomide and a specific peptide as active ingredients.
  • the method for administering the antitumor agent of the present invention is not particularly limited, and can be selected according to use. For example, surgical treatment such as oral administration, intravenous administration, and indwelling can be exemplified.
  • temozolomide is generally administered orally and is preferably administered orally.
  • the antitumor agent of the present invention is a combination of drugs each containing temozolomide and a specific peptide as active ingredients
  • a first container containing temozolomide and a second container containing a specific peptide And the aspect provided with these is mentioned.
  • the first container and the second container are not particularly limited as long as each drug can be stored independently.
  • seat in which the part which accommodates a temozolomide, and the part which accommodates a specific peptide may be sufficient.
  • the glioma treatment method includes administering a peptide (specific peptide) which is the following (a) or (b) to a patient.
  • a peptide specifically peptide
  • B A peptide comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted or added in the amino acid sequence (a) and having an antiglioma action.
  • the specific peptide is (b): a peptide in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a), the number of amino acid residues to be deleted, substituted or added is the specific peptide If it is a range which has an antiglioma action, it will not restrict
  • the number is 1 to 9, preferably 1 to 6, and more preferably 1 to 4.
  • the total number of amino acid residues of the peptide (b) is preferably 30 or less, more preferably 28 or less, and 25 or less. More preferred.
  • a specific peptide having the above amino acid sequence is effective for the treatment of glioma.
  • treatment includes not only eliminating or reducing symptoms caused by glioma but also suppressing the degree of progression of symptoms.
  • the “anti-glioma action” includes an action to suppress glioma growth, migration, infiltration and the like in addition to an action to eliminate or reduce glioma.
  • the method for administering the specific peptide to the patient is not particularly limited, and examples thereof include oral administration, intravenous administration, and surgical treatment.
  • the amount of the specific peptide administered to the patient is not particularly limited, and can be selected according to the state of the tumor, the type and amount of other components used together with the specific peptide, and the like.
  • the glioma treatment method of the present invention may be a single use of a drug containing a specific peptide as an active ingredient, or may be used in combination with another drug such as temozolomide.
  • glioblastoma Human glioblastoma (glioblastoma) cell line T98G was cultured in RPMI 1640 containing 10% FBS at 37 ° C. and 5% CO 2 in an incubator. 0.15% NaHCO 3 , 2 mM L-glutamine, and Penicillin-Streptomycin Solution (Wako Pure Chemical Industries, Ltd.) were added to the medium.
  • T98G cells cultured in a 96-well plate coated with FN (0.25 ⁇ g / mL) were seeded with a predetermined amount of PDGF (Wako Pure Chemical Industries, Ltd.), TNIIIA2 (SEQ ID NO: 2) and FNIII14 (SEQ ID NO: 1), 37 ° C., and cultured in an incubator at a 5% CO 2 condition. Thereafter, the number of viable cells was measured using Cell Counting Kit-8 (Dojindo Laboratories). Multimode Plate Reader ARVO TM X4 (Perkin Elmer) was used for the absorbance measurement.
  • the results are shown in FIG.
  • the numbers in the figure indicate the amount of TNIIIA2 added ( ⁇ g / mL).
  • ⁇ PDGF represents a group to which PDGF was not added
  • + PDGF represents a group to which 10 ng / mL of PDGF was added.
  • + FNIII14 25 represents a group to which FNIII14 was added at 25 ⁇ g / mL
  • + FNIII14 50 represents a group to which FNIII14 was added at 50 ⁇ g / mL.
  • the inhibitory effect on anchorage-independent cell proliferation by specific peptides was evaluated by soft agar colony assay.
  • the anchorage-independent cell proliferative ability is an index of cell canceration, and the more proliferating, the more marked the canceration of cells.
  • a 1.4% agarose gel (Bacto Agar, BD) was sterilized and dissolved in an autoclave and then brought to 45 ° C. in a thermostatic bath.
  • a double-concentration medium containing 20% FBS at 45 ° C. was mixed with an agarose gel in an equal amount, and 1 mL was added to a 12-well plate and solidified at room temperature.
  • the agarose gel mixed in an equal amount as described above was further mixed in an equal amount with the reagent and the suspension of T98G cells cultured by the method described in Example 1, and 1 mL was added to each well. After allowing to stand at 4 ° C. for 8 minutes to solidify, 1 mL of medium containing 10% FBS was added to each well. The cells were cultured in an incubator for a certain period under conditions of 37 ° C. and 5% CO 2 , and then the number of colonies was counted.
  • control is a group in which no reagent is added
  • TNIIIA2 is a group in which TNIIIA2 is added at 25 ⁇ g / mL
  • PDGF is a group in which PDGF is added at 10 ng / mL
  • TNIIIA2 + PDGF is TNIIIA2 at 25 ⁇ g / mL.
  • TNIIIA2 enhanced the anchorage-independent growth of T98G cells, and it was found that the enhancement effect was more remarkable when PDGF was added. Furthermore, it was found that when FNIII14 was added, the anchorage-independent growth of T98G cells was remarkably suppressed, and FNIII14 suppressed the promotion of anchorage-independent growth by TNIIIA2.
  • Example 3 Evaluation of tumor cell dispersion inhibitory effect> The inhibitory effect of the specific peptide on tumor cell dispersion was evaluated by scattering assay. It means that the more the cells are dispersed, the more the tumor moves and invades.
  • T98G cells cultured by the method described in Example 1 were seeded on a plate coated with FN (0.25 ⁇ g / mL), and cultured in an incubator under conditions of 37 ° C. and 5% CO 2 . Each reagent was allowed to react with the cells increased in a paving stone shape, and fixed and stained with Diff-Quik (Sysmex Corporation) after a certain time, and the morphological changes of the cells were observed. The morphological changes of the cells were photographed with Motic Image Plus 2.2S (Shimadzu Rika Co., Ltd.).
  • control is a group to which no reagent was added
  • TNIIIA2 was a group to which TNIIIA2 was added at 25 ⁇ g / mL
  • PDGF was a group to which PDGF was added at 10 ng / mL
  • TNIIIA2 + PDGF was TNIIIA2 at 25 ⁇ g / mL.
  • TNIIIA2 induces the dispersion of T98G cells. Furthermore, TNIIIA2 was found to induce T98G cell dispersion regardless of the addition or absence of PDGF. On the other hand, as shown in the photograph on the left side of FIG. 3, when FNIII14 was added, even when TNIIIA2 was added, no dispersion of T98G cells was observed, indicating that FNIII14 inhibits the induction of cell dispersion by TNIIIA2.
  • Example 4 mouse side back The inhibitory effect of specific peptides on tumor formation was evaluated by animal experiments using mice.
  • a rat glioma cell line 9L (5 ⁇ 10 5 cells / 100 ⁇ L) was transplanted subcutaneously into the left back of a 6-week-old male nude mouse (Balb / c nu / nu).
  • the mice were randomly divided into two groups.
  • One group contained FNIII14 in which the second-position glutamic acid was converted to D-form in order to prevent degradation in vivo.
  • Groups were infused with a mutant of FNIII14 (SEQ ID NO: 3) inactivated by shuffling the sequence in the tail vein daily for 21 days in an amount of 250 ⁇ g per individual.
  • Mouse weights were also measured every other day.
  • control peptide is a control group to which a mutant of FNIII14 was administered
  • D-Glu FNIII14 was a tumor volume (mm 3 ) of a group to which FNIII14 in which glutamic acid at the second position was converted to D-form was administered
  • Example 5 Evaluation of temozolomide activity enhancing effect> The enhancement effect of temozolomide activity by specific peptides was evaluated by the following test. T98G cells cultured by the method described in Example 1 were seeded on a plate coated with FN (0.25 ⁇ g / mL), and cultured in an incubator under conditions of 37 ° C. and 5% CO 2 . The cell viability was examined by allowing temozolomide alone or a combination of temozolomide and FNIII14 to act on the cultured cells at varying concentrations.
  • Temozolomide indicates a change in cell viability of a group in which temozolomide alone was allowed to act
  • the combination index (CI) was calculated by the following formula.
  • a CI value of less than 1 means that a synergistic effect is exhibited by the combined use of temozolomide and FNIII14.
  • the survival rate of the cells treated with the combination of temozolomide and FNIII14 was lower than that of the group treated with temozolomide alone, indicating that FNIII14 enhances the activity of temozolomide. Furthermore, the value of CI when temozolomide and FNIII14 were used in combination was smaller than 1, indicating that a synergistic effect appeared.
  • FNIII14 (TEATITGLEPPGTEYTIVIAL, SEQ ID NO: 1), TNIIIA2 (RSTDLPGLKAATYYTITIRGVC, SEQ ID NO: 2), and FNIII14 mutant (TEATITGLEPGTTETAYVAALC, SEQ ID NO: 3) were purchased from Operon Biotechnology Co., Ltd.
  • D-Glu FNIII14 T- [D-Glu] -ATITGLEPGTTYTIVIAL
  • T98G Human glioblastoma cell line
  • Rat glioma cell line 9L was provided by the Department of Neurosurgery, School of Medicine, University of Tsukuba.

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Abstract

　La présente invention se rapporte à un inhibiteur de la croissance cellulaire dépendant de PDGF qui comprend, en tant que composant actif, un peptide comprenant une séquence d'acides aminés représentée par TEATITGLEPGTEYTIYVIAL, ou un peptide comprenant une séquence d'acides aminés dans lequel la séquence d'acides aminés mentionnée ci-dessus comporte au moins un résidu d'acide aminé supprimé, substitué ou ajouté. L'invention concerne également un inhibiteur de la dispersion cellulaire, un renforçateur de l'activité du témozolomide et un agent antitumoral.
PCT/JP2015/067651 2014-06-18 2015-06-18 Inhibiteur de la croissance cellulaire dépendant de pdgf, procédé d'inhibition de la croissance cellulaire dépendant de pdgf, inhibiteur de la dispersion des cellules, procédé d'inhibition de la dispersion des cellules, renforçateur de l'activité du témozolomide, et agent antitumoral WO2015194643A1 (fr)

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JP2019006732A (ja) * 2017-06-27 2019-01-17 学校法人東京理科大学 Ｍｙｃタンパク質阻害剤、Ｍｙｃタンパク質阻害方法、抗神経芽腫剤及びＭｙｃタンパク質の過剰発現を伴う疾患の治療方法
WO2021132539A1 (fr) * 2019-12-27 2021-07-01 株式会社 ブルボン Régulateur de croissance cellulaire

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019006732A (ja) * 2017-06-27 2019-01-17 学校法人東京理科大学 Ｍｙｃタンパク質阻害剤、Ｍｙｃタンパク質阻害方法、抗神経芽腫剤及びＭｙｃタンパク質の過剰発現を伴う疾患の治療方法
WO2021132539A1 (fr) * 2019-12-27 2021-07-01 株式会社 ブルボン Régulateur de croissance cellulaire

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