WO2015194643A1 - Pdgf-dependent cell-growth inhibitor, pdgf-dependent cell-growth inhibiting method, cell dispersion inhibitor, cell dispersion inhibiting method, temozolomide activity enhancer, and antitumor agent - Google Patents

Pdgf-dependent cell-growth inhibitor, pdgf-dependent cell-growth inhibiting method, cell dispersion inhibitor, cell dispersion inhibiting method, temozolomide activity enhancer, and antitumor agent 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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French (fr)
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/en
Publication of WO2015194643A1 publication Critical patent/WO2015194643A1/en

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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, 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.

Abstract

　Provided is a PDGF-dependent cell-growth inhibitor that includes, as an active component, a peptide comprising an amino acid sequence represented by TEATITGLEPGTEYTIYVIAL, or a peptide comprising an amino acid sequence in which the aforementioned amino acid sequence has at least one amino acid residue deleted, substituted, or added. Also provided are a cell dispersion inhibitor, a temozolomide activity enhancer, and an antitumor agent.

Description

ＰＤＧＦ依存性細胞増殖抑制剤、ＰＤＧＦ依存性細胞増殖の抑制方法、細胞分散抑制剤、細胞分散の抑制方法、テモゾロミド活性増強剤及び抗腫瘍剤PDGF-dependent cell growth inhibitor, PDGF-dependent cell growth suppression method, cell dispersion inhibitor, cell dispersion suppression method, temozolomide activity enhancer, and antitumor agent
　本発明は、ＰＤＧＦ依存性細胞増殖抑制剤、ＰＤＧＦ依存性細胞増殖の抑制方法、細胞分散抑制剤、細胞分散の抑制方法、テモゾロミド活性増強剤及び抗腫瘍剤に関する。 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.
　グリア細胞（膠細胞）由来の脳腫瘍である神経膠腫は極めて悪性度が高く、治癒率は１０％に満たない。その理由として、神経膠腫細胞の高い増殖能ととともに、活発な移動・浸潤能が腫瘍の外科的な摘出を困難にしていることが挙げられる。従って、腫瘍の外科的な摘出を困難にする腫瘍細胞の増殖及び移動・浸潤を有効に抑制することができれば外科的な治療が可能となり、治癒率の向上が期待できる。また、既存の薬剤の効果を有効に増強することができれば治癒率の向上、副作用の低減等の効果が期待できる。 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.
　腫瘍の増大や浸潤を抑制する手段として、細胞外マトリクスであるフィブロネクチン由来のアミノ酸配列からなり、細胞接着阻害活性を有するペプチドが癌転移抑制剤として有用であるとの報告がなされている（例えば、特許文献１及び２）。また、フィブロネクチン由来のアミノ酸配列からなるペプチドが既存の抗がん剤の活性増強剤として有用であるとの報告がなされている（例えば、特許文献３）。 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).
特開平１０－１４７６００号公報Japanese Patent Laid-Open No. 10-147600 特開２０００－２６４９００号公報JP 2000-264900 A 国際公開第２０１２／１２４６４１号International Publication No. 2012/1244641
　特許文献１及び２では細胞接着阻害活性を有するペプチドの癌転移抑制剤としての実施例による効果の実証はなされていない。また、特許文献３ではフィブロネクチン由来のアミノ酸配列からなるペプチドと、神経膠腫の治療薬として用いられるテモゾロミドとの併用については検討されていない。本発明者らは上記状況に鑑み、腫瘍細胞の増殖又は分散の抑制に有用な、ＰＤＧＦ依存性細胞増殖抑制剤、ＰＤＧＦ依存性細胞増殖の抑制方法、細胞分散抑制剤、細胞分散の抑制方法、テモゾロミド活性増強剤及び抗腫瘍剤を提供することを目的とする。 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. In view of the above circumstances, 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.
　前記課題を達成するための具体的手段は以下のとおりである。
＜１＞以下の（ａ）又は（ｂ）であるペプチドを有効成分として含む、ＰＤＧＦ依存性細胞増殖抑制剤。
（ａ）Ｔｈｒ－Ｇｌｕ－Ａｌａ－Ｔｈｒ－Ｉｌｅ－Ｔｈｒ－Ｇｌｙ－Ｌｅｕ－Ｇｌｕ－Ｐｒｏ－Ｇｌｙ－Ｔｈｒ－Ｇｌｕ－Ｔｙｒ－Ｔｈｒ－Ｉｌｅ－Ｔｙｒ－Ｖａｌ－Ｉｌｅ－Ａｌａ－Ｌｅｕ（配列番号１）で表されるアミノ酸配列からなるペプチド。
（ｂ）アミノ酸配列（ａ）において１若しくは数個のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなり、ＰＤＧＦ依存性細胞増殖抑制作用を有するペプチド。 Specific means for achieving the above object are as follows.
<1> 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.
(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.
＜２＞前記細胞はＰＤＧＦ受容体を発現する、肉腫及び癌腫からなる群より選択される少なくとも１種の細胞である＜１＞に記載のＰＤＧＦ依存性細胞増殖抑制剤。 <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＞前記細胞は神経膠腫、繊維肉腫、骨肉腫、乳がん及び肺がんからなる群より選択される少なくとも１種の細胞である、＜１＞又は＜２＞に記載のＰＤＧＦ依存性細胞増殖抑制剤。 <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＞＜１＞～＜３＞のいずれか１項に記載のＰＤＧＦ依存性細胞増殖抑制剤を細胞に接触させることを含む、ＰＤＧＦ依存性細胞増殖の抑制方法。 <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>.
＜５＞以下の（ａ）又は（ｂ）であるペプチドを有効成分として含む、細胞分散抑制剤。
（ａ）Ｔｈｒ－Ｇｌｕ－Ａｌａ－Ｔｈｒ－Ｉｌｅ－Ｔｈｒ－Ｇｌｙ－Ｌｅｕ－Ｇｌｕ－Ｐｒｏ－Ｇｌｙ－Ｔｈｒ－Ｇｌｕ－Ｔｙｒ－Ｔｈｒ－Ｉｌｅ－Ｔｙｒ－Ｖａｌ－Ｉｌｅ－Ａｌａ－Ｌｅｕ（配列番号１）で表されるアミノ酸配列からなるペプチド。
（ｂ）アミノ酸配列（ａ）において１若しくは数個のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなり、細胞分散抑制作用を有するペプチド。 <5> 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＞前記細胞は細胞間接着によって形成された集団又は集隗からの細胞の解離が生じる、肉腫及び癌腫からなる群より選択される少なくとも１種の細胞である＜５＞に記載の細胞分散抑制剤。 <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＞前記細胞は神経膠腫、繊維肉腫、骨肉腫、乳がん及び肺がんからなる群より選択される少なくとも１種の細胞である、＜５＞又は＜６＞に記載の細胞分散抑制剤。 <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＞＜５＞～＜７＞のいずれか１項に記載の細胞分散抑制剤を細胞に接触させることを含む、細胞分散の抑制方法。 <8> A method for inhibiting cell dispersion, comprising contacting the cell dispersion inhibitor according to any one of <5> to <7> with cells.
＜９＞以下の（ａ）又は（ｂ）であるペプチドを有効成分として含む、テモゾロミド活性増強剤。
（ａ）Ｔｈｒ－Ｇｌｕ－Ａｌａ－Ｔｈｒ－Ｉｌｅ－Ｔｈｒ－Ｇｌｙ－Ｌｅｕ－Ｇｌｕ－Ｐｒｏ－Ｇｌｙ－Ｔｈｒ－Ｇｌｕ－Ｔｙｒ－Ｔｈｒ－Ｉｌｅ－Ｔｙｒ－Ｖａｌ－Ｉｌｅ－Ａｌａ－Ｌｅｕ（配列番号１）で表されるアミノ酸配列からなるペプチド。
（ｂ）アミノ酸配列（ａ）において１若しくは数個のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなり、テモゾロミド活性増強作用を有するペプチド。 <9> 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).
＜１０＞テモゾロミドと、以下の（ａ）又は（ｂ）であるペプチドと、を有効成分として含む抗腫瘍剤。
（ａ）Ｔｈｒ－Ｇｌｕ－Ａｌａ－Ｔｈｒ－Ｉｌｅ－Ｔｈｒ－Ｇｌｙ－Ｌｅｕ－Ｇｌｕ－Ｐｒｏ－Ｇｌｙ－Ｔｈｒ－Ｇｌｕ－Ｔｙｒ－Ｔｈｒ－Ｉｌｅ－Ｔｙｒ－Ｖａｌ－Ｉｌｅ－Ａｌａ－Ｌｅｕ（配列番号１）で表されるアミノ酸配列からなるペプチド。
（ｂ）アミノ酸配列（ａ）において１若しくは数個のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなり、テモゾロミド活性増強作用を有するペプチド。 <10> An antitumor agent comprising temozolomide and the following 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) 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).
　本発明によれば、腫瘍細胞の増殖又は分散の抑制に有用な、ＰＤＧＦ依存性細胞増殖抑制剤、ＰＤＧＦ依存性細胞増殖の抑制方法、細胞分散抑制剤、細胞分散の抑制方法、テモゾロミド活性増強剤及び抗腫瘍剤が提供される。 According to the present invention, 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.
特定ペプチドによるＰＤＧＦ依存性細胞増殖抑制効果の評価を示す図である。It is a figure which shows evaluation of the PDGF dependence cell growth inhibitory effect by a specific peptide. 特定ペプチドによる足場非依存性細胞増殖抑制効果の評価を示す図である。It is a figure which shows evaluation of the anchorage independent cell growth inhibitory effect by a specific peptide. 特定ペプチドによる腫瘍細胞分散抑制効果の評価を示す図である。It is a figure which shows evaluation of the tumor cell dispersion | distribution inhibitory effect by a specific peptide. 特定ペプチドによるマウス側背部における腫瘍形成抑制効果の評価を示す図である。It is a figure which shows evaluation of the tumor formation inhibitory effect in the mouse | mouth side back part by a specific peptide. 特定ペプチドによるテモゾロミド活性増強効果の評価を示す図である。It is a figure which shows evaluation of the temozolomide activity enhancement effect by a specific peptide.
　以下、本発明の実施の形態について説明する。これらの説明及び実施例は本発明を例示するものであり、本発明の範囲を制限するものではない。
　本明細書において「～」を用いて示された数値範囲は、「～」の前後に記載される数値をそれぞれ最小値及び最大値として含む範囲を示す。アミノ酸配列の記載は左側がＮ末端側であり、アミノ酸残基は当該技術分野で周知の一文字表記（例えば、グリシン残基を「Ｇ」）または三文字表記（例えば、グリシン残基を「Ｇｌｙ」）で表記する場合がある。 Embodiments of the present invention will be described below. These descriptions and examples are illustrative of the invention and are not intended to limit the scope of the invention.
In the present specification, 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. In the description of the amino acid sequence, the left side is the N-terminal side, and 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.
＜ＰＤＧＦ依存性細胞増殖抑制剤＞
　本発明のＰＤＧＦ依存性細胞増殖抑制剤は、以下の（ａ）又は（ｂ）であるペプチド（以下、特定ペプチドともいう）を有効成分として含む。
（ａ）Ｔｈｒ－Ｇｌｕ－Ａｌａ－Ｔｈｒ－Ｉｌｅ－Ｔｈｒ－Ｇｌｙ－Ｌｅｕ－Ｇｌｕ－Ｐｒｏ－Ｇｌｙ－Ｔｈｒ－Ｇｌｕ－Ｔｙｒ－Ｔｈｒ－Ｉｌｅ－Ｔｙｒ－Ｖａｌ－Ｉｌｅ－Ａｌａ－Ｌｅｕ（配列番号１）で表されるアミノ酸配列からなるペプチド。
（ｂ）アミノ酸配列（ａ）において１若しくは数個のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなり、かつＰＤＧＦ依存性細胞増殖抑制作用を有するペプチド。 <PDGF-dependent cell growth inhibitor>
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.
　特定ペプチドが（ｂ）：アミノ酸配列（ａ）において１若しくは数個のアミノ酸が欠失、置換若しくは付加されてなるペプチドである場合、欠失、置換若しくは付加されるアミノ酸残基の数は特定ペプチドがＰＤＧＦ依存性細胞増殖抑制作用を有する範囲であれば特に制限されない。例えば、１～９個であり、１～６個であることが好ましく、１～４個であることがより好ましい。合成の効率、取扱性、安定性等の観点からは、（ｂ）のペプチドのアミノ酸残基の総数は３０以下であることが好ましく、２８以下であることがより好ましく、２５以下であることがより好ましい。 When 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. For example, the number is 1 to 9, preferably 1 to 6, and more preferably 1 to 4. From the viewpoint of synthesis efficiency, handleability, stability, etc., 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.
　上記（ａ）であるペプチドのアミノ酸配列は、細胞外マトリックスタンパク質分子の一つであるフィブロネクチンを構成するフィブロネクチンＩＩＩ型ドメインに由来するものである。本発明者らは、上記アミノ酸配列を有するペプチドがＰＤＧＦ依存性の細胞増殖を抑制する作用を有することを見出した。なお、「ＰＤＧＦ依存性」とは細胞の増殖が血小板由来増殖因子（ＰＤＧＦ）受容体の発現を伴っていることを意味する。ＰＤＧＦが発現しているか否かは、抗ＰＤＧＦ受容体抗体を用いてウェスタンブロッティング、フローサイトメトリー等によって確認することができる。 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.
　本発明のＰＤＧＦ依存性細胞増殖抑制剤がＰＤＧＦ依存性の細胞増殖を抑制する作用を有する理由は明らかではないが、以下のように推測している。すなわち、ＰＤＧＦ受容体の発現が認められる腫瘍では、炎症時や病変時に一時的に発現する性質を有する細胞外マトリクスであるテネイシンＣの発現が著しく上昇する。テネイシンＣより遊離したペプチド（ＴＮＩＩＩＡ２）は細胞接着分子であるインテグリンを活性化する作用を有しており、これによって腫瘍細胞のＰＤＧＦ依存性増殖が増強される。他方、本発明のＰＤＧＦ依存性細胞増殖抑制剤に含まれる特定ペプチドはインテグリンを不活性化する作用を有している。その結果、ＴＮＩＩＩＡ２のインテグリン活性化作用が抑制され、腫瘍細胞の増殖が抑制されると考えられる。ＴＮＩＩＩＡ２についてはＪ．Ｂｉｏｌ．Ｃｈｅｍ．，Ｖｏｌ．２８２、ｐｐ．３４９２９－３４９３７，（２００７）等に詳細な記載がある。 The reason why the PDGF-dependent cell growth inhibitor of the present invention has an action of suppressing PDGF-dependent cell growth is not clear, but is presumed as follows. 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. On the other hand, the specific peptide contained in the PDGF-dependent cell growth inhibitor of the present invention has an effect of inactivating integrin. As a result, it is considered that the integrin activation action of TNIIIA2 is suppressed and the growth of tumor cells is suppressed. For TNIIIA2, see J.A. Biol. Chem. , Vol. 282, pp. No. 34929-34937, (2007), and the like.
　ＰＤＧＦ依存性の細胞増殖を伴う腫瘍として具体的には、神経膠腫、繊維肉腫や骨肉腫等の肉腫、乳がんや肺がん等の癌種などが挙げられる。神経膠腫としては星細胞系腫瘍に分類される毛様細胞性星細胞腫、びまん性星細胞腫、退形成性星細胞腫及び神経膠芽腫、乏突起膠腫、上衣腫、並びに脈絡叢乳頭腫が挙げられる。ＰＤＧＦの発現の程度は本発明の効果が得られる限り特に制限されないが、ＰＤＧＦをより強く発現する腫瘍において本発明の効果がより顕著である。例えば、神経膠腫の中でも悪性度が高い神経膠芽腫はＰＤＧＦを高発現することが知られており、本発明の効果が特に顕著である。 Specific examples of 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. For example, 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.
　本発明のＰＤＧＦ依存性細胞増殖抑制剤に含まれる特定ペプチドは、用途に応じて種々の改変を施してもよい。例えば、ポリエチレングリコール等の水溶性ポリマー、硫酸基、水溶性アミノ酸などを特定ペプチドの末端に連結して水溶性を向上させたり、多量体化したりしてもよい。また、特定ペプチドを構成するアミノ酸残基のそれぞれは本発明の効果が達成される限りＬ体又はＤ体のいずれであってもよい。体内での特定ペプチドの分解を抑制する観点からは、アミノ酸配列の少なくとも１部（例えば、第２位のＧｌｕ）をＤ体とすることが好ましい。特定ペプチドの作製方法は特に制限されず、遺伝子工学的方法又は有機合成化学的方法のいずれであってもよい。 The specific peptide contained in the PDGF-dependent cell growth inhibitor of the present invention may be variously modified depending on the application. For example, 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. In addition, 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.
　本発明のＰＤＧＦ依存性細胞増殖抑制剤は、使用態様に応じて特定ペプチド以外の成分を含んでいてもよい。特定ペプチド以外の成分としては、薬剤の調製に一般に用いられる媒質及び製剤用添加物を挙げることができる。媒質及び製剤用添加物の種類は、特に制限されない。媒質としては、固体媒質（例えば、ゼラチン、乳糖）及び液体媒質（例えば、アルコール、水、生理食塩水）が挙げられる。製剤用添加物としては、賦形剤、崩壊剤、結合剤、滑沢剤、界面活性剤、緩衝剤、溶解補助剤、安定化剤、等張化剤などが挙げられる。 The PDGF-dependent cell growth inhibitor of the present invention may contain components other than the specific peptide depending on the use mode. Examples of 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. Examples of the medium include a solid medium (for example, gelatin and lactose) and a liquid medium (for example, alcohol, water, and physiological saline). Examples of the additive for preparation include excipients, disintegrants, binders, lubricants, surfactants, buffers, solubilizers, stabilizers, tonicity agents, and the like.
　本発明のＰＤＧＦ依存性細胞増殖抑制剤の形態は特に制限されず、用途に応じて選択できる。例えば、錠剤、顆粒剤、散剤、カプセル剤、懸濁剤、シロップ、乳剤、リモナーデ剤等の経口投与に適した形態、注射用アンプル剤、注射用凍結乾燥粉末剤、経肺投与用乾燥粉末剤などが挙げられる。 The form of the PDGF-dependent cell growth inhibitor of the present invention is not particularly limited, and can be selected according to use. For example, 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.
＜ＰＤＧＦ依存性細胞増殖の抑制方法＞
　本発明のＰＤＧＦ依存性細胞増殖の抑制方法は、本発明のＰＤＧＦ依存性細胞増殖抑制剤を細胞に接触させることを含む。「細胞に接触させること」とは、ＰＤＧＦ依存性の増殖を示す細胞と、本発明のＰＤＧＦ依存性細胞増殖抑制剤に含まれる特定ペプチドとが接触する状態にすることを意味し、ＰＤＧＦ依存性の増殖を示す細胞から構成される腫瘍やがん組織に接触させることを含む。ＰＤＧＦ依存性細胞増殖抑制剤を細胞に接触させる方法は特に制限されず、経口投与、静脈内投与、留置等の外科的処置等を挙げることができる。細胞に接触させるＰＤＧＦ依存性細胞増殖抑制剤の量は特に制限されず、細胞の状態、特定ペプチドとともに使用する他の成分の種類や量等に応じて選択できる。 <Method for inhibiting PDGF-dependent cell proliferation>
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.
＜細胞分散抑制剤＞
　本発明の細胞分散抑制剤は、以下の（ａ）又は（ｂ）であるペプチド（特定ペプチド）を有効成分として含む。
（ａ）Ｔｈｒ－Ｇｌｕ－Ａｌａ－Ｔｈｒ－Ｉｌｅ－Ｔｈｒ－Ｇｌｙ－Ｌｅｕ－Ｇｌｕ－Ｐｒｏ－Ｇｌｙ－Ｔｈｒ－Ｇｌｕ－Ｔｙｒ－Ｔｈｒ－Ｉｌｅ－Ｔｙｒ－Ｖａｌ－Ｉｌｅ－Ａｌａ－Ｌｅｕ（配列番号１）で表されるアミノ酸配列からなるペプチド。
（ｂ）アミノ酸配列（ａ）において１若しくは数個のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ細胞分散抑制作用を有するペプチド。 <Cell dispersion inhibitor>
The cell dispersion inhibitor of the present invention contains the following peptide (specific 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.
　特定ペプチドが（ｂ）：アミノ酸配列（ａ）において１若しくは数個のアミノ酸が欠失、置換若しくは付加されてなるペプチドである場合、欠失、置換若しくは付加されるアミノ酸残基の数は特定ペプチドが細胞分散抑制作用を有する範囲であれば特に制限されない。例えば、１～９個であり、１～６個であることが好ましく、１～４個であることがより好ましい。合成の効率、取扱性、安定性等の観点からは、（ｂ）のペプチドのアミノ酸残基の総数は３０以下であることが好ましく、２８以下であることがより好ましく、２５以下であることがより好ましい。 When 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 | distribution inhibitory effect, it will not restrict | limit in particular. For example, the number is 1 to 9, preferably 1 to 6, and more preferably 1 to 4. From the viewpoint of synthesis efficiency, handleability, stability, etc., 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.
　本発明者らは、上記アミノ酸配列を有する特定ペプチドが細胞の分散を抑制する作用を有することを見出した。腫瘍を形成する細胞の分散が抑制されることで、腫瘍の移動や浸潤が有効に抑制される。その結果、外科的な摘出等の手法の適用が可能となり、治療方法の選択の幅が広がる。なお、細胞の「分散」とは、細胞同士の細胞間接着によって形成された集団又は集隗状態から個々の細胞が解離することを意味する。悪性化した腫瘍細胞は、「分散」することで原発腫瘍組織から解離し、周辺組織へ浸潤・転移する。 The present inventors have found that the specific peptide having the amino acid sequence has an action of suppressing cell dispersion. 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.
　本発明の細胞分散抑制剤が細胞の分散を抑制する作用を有する理由は明らかではないが、以下のように推測している。すなわち、細胞の分散は腫瘍部位に発現したテネイシンＣより遊離したペプチド（ＴＮＩＩＩＡ２）のインテグリン活性化作用により誘導される。従って、インテグリンを不活性化する作用を有する特定ペプチドを含む本発明の細胞分散抑制剤を適用することにより、細胞の分散が抑制されると考えられる。 The reason why the 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.
　細胞の分散が認められる腫瘍として具体的には、神経膠腫、繊維肉腫や骨肉腫等の肉腫、乳がんや肺がん等の癌種などが挙げられる。神経膠腫としては星細胞系腫瘍に分類される毛様細胞性星細胞腫、びまん性星細胞腫、退形成性星細胞腫及び神経膠芽腫、乏突起膠腫、上衣腫、並びに脈絡叢乳頭腫が挙げられる。細胞の分散の程度は本発明の効果が得られる限り特に制限されないが、細胞の分散がより活発である腫瘍において本発明の効果がより顕著である。例えば、神経膠腫の中でも悪性度が高い神経膠芽腫は細胞の分散が活発であることが知られており、本発明の効果が特に顕著である。 Specific examples of 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. For example, glioblastoma having high malignancy among gliomas is known to have active cell dispersion, and the effect of the present invention is particularly remarkable.
　本発明の細胞分散抑制剤に含まれる特定ペプチドは、用途に応じて種々の改変を施してもよい。例えば、ポリエチレングリコール等の水溶性ポリマー、硫酸基、水溶性アミノ酸などを特定ペプチドの末端に連結して水溶性を向上させたり、多量体化したりしてもよい。また、特定ペプチドを構成するアミノ酸残基のそれぞれは本発明の効果が達成される限りＬ体又はＤ体のいずれであってもよい。体内での特定ペプチドの分解を抑制する観点からは、アミノ酸配列の少なくとも１部（例えば、第２位のＧｌｕ）をＤ体とすることが好ましい。特定ペプチドの作製方法は特に制限されず、遺伝子工学的方法又は有機合成化学的方法のいずれであってもよい。 The specific peptide contained in the cell dispersion inhibitor of the present invention may be variously modified depending on the application. For example, 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. In addition, 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.
　本発明の細胞分散抑制剤は、使用態様に応じて特定ペプチド以外の成分を含んでいてもよい。特定ペプチド以外の成分としては、薬剤の調製に一般に用いられる媒質及び製剤用添加物を挙げることができる。媒質及び製剤用添加物の種類は、特に制限されない。媒質としては、固体媒質（例えば、ゼラチン、乳糖）及び液体媒質（例えば、アルコール、水、生理食塩水）が挙げられる。製剤用添加物としては、賦形剤、崩壊剤、結合剤、滑沢剤、界面活性剤、緩衝剤、溶解補助剤、安定化剤、等張化剤などが挙げられる。 The cell dispersion inhibitor of the present invention may contain components other than the specific peptide depending on the use mode. Examples of 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. Examples of the medium include a solid medium (for example, gelatin and lactose) and a liquid medium (for example, alcohol, water, and physiological saline). Examples of the additive for preparation include excipients, disintegrants, binders, lubricants, surfactants, buffers, solubilizers, stabilizers, tonicity agents, and the like.
　本発明の細胞分散抑制剤の形態は特に制限されず、用途に応じて選択できる。例えば、錠剤、顆粒剤、散剤、カプセル剤、懸濁剤、シロップ、乳剤、リモナーデ剤等の経口投与に適した形態、注射用アンプル剤、注射用凍結乾燥粉末剤、経肺投与用乾燥粉末剤などが挙げられる。 The form of the cell dispersion inhibitor of the present invention is not particularly limited and can be selected depending on the application. For example, 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.
＜細胞分散の抑制方法＞
　本発明の細胞分散の抑制方法は、本発明の細胞分散抑制剤を細胞に接触させることを含む。「細胞に接触させること」とは、細胞間接着によって集団若しくは集隗を形成している細胞、又は集団若しくは集隗から解離している細胞と、本発明の細胞分散抑制剤に含まれる特定ペプチドとが接触する状態にすることを意味し、集団又は集隗からの細胞の解離が未だ認められないが解離が生じる可能性が大きい状態、又は解離がすでに認められる状態の腫瘍やがん組織に接触させることを含む。細胞分散抑制を細胞に接触させる方法は特に制限されず、経口投与、静脈内投与、留置等の外科的処置等を挙げることができる。細胞に接触させる細胞分散抑制剤の量は特に制限されず、細胞の状態、特定ペプチドとともに使用する他の成分の種類や量等に応じて選択できる。 <Method for inhibiting cell dispersion>
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.
＜テモゾロミド活性増強剤＞
　本発明のテモゾロミド活性増強剤は、以下の（ａ）又は（ｂ）であるペプチド（特定ペプチド）を有効成分として含む。
（ａ）Ｔｈｒ－Ｇｌｕ－Ａｌａ－Ｔｈｒ－Ｉｌｅ－Ｔｈｒ－Ｇｌｙ－Ｌｅｕ－Ｇｌｕ－Ｐｒｏ－Ｇｌｙ－Ｔｈｒ－Ｇｌｕ－Ｔｙｒ－Ｔｈｒ－Ｉｌｅ－Ｔｙｒ－Ｖａｌ－Ｉｌｅ－Ａｌａ－Ｌｅｕ（配列番号１）で表されるアミノ酸配列からなるペプチド。
（ｂ）アミノ酸配列（ａ）において１若しくは数個のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなり、かつテモゾロミド活性増強作用を有するペプチド。 <Temozolomide activity enhancer>
The temozolomide activity enhancer of the present invention contains the following peptide (specific 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 temozolomide activity enhancing action.
　特定ペプチドが（ｂ）：アミノ酸配列（ａ）において１若しくは数個のアミノ酸が欠失、置換若しくは付加されてなるペプチドである場合、欠失、置換若しくは付加されるアミノ酸残基の数は特定ペプチドがテモゾロミド活性増強作用を有する範囲であれば特に制限されない。例えば、１～９個であり、１～６個であることが好ましく、１～４個であることがより好ましい。合成の効率、取扱性、安定性等の観点からは、（ｂ）のペプチドのアミノ酸残基の総数は３０以下であることが好ましく、２８以下であることがより好ましく、２５以下であることがより好ましい。 When 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. For example, the number is 1 to 9, preferably 1 to 6, and more preferably 1 to 4. From the viewpoint of synthesis efficiency, handleability, stability, etc., 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.
　本発明のテモゾロミド活性増強剤は、テモゾロミド（３，４－ジヒドロ－３－メチル－４－オキソイミダゾ［５，１－ｄ］［１，２，３，５］テトラジン－８－カルボキサミド）の抗腫瘍作用を増強する。テモゾロミドはアルキル化剤に属する経口薬であり、分子量が小さく血液脳関門を通過できるために脳腫瘍の治療薬として使用されている。 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.
　本発明者らは、上記アミノ酸配列を有する特定ペプチドがテモゾロミドの抗腫瘍作用を増強することを見出した。すなわち、特定ペプチドを有効成分として含む本発明のテモゾロミド活性増強剤をテモゾロミドと併用することにより、テモゾロミド又は特定ペプチドを単独で使用した場合を上回る抗腫瘍効果を得ることができる。また、本発明のテモゾロミド活性増強剤をテモゾロミドと併用することで充分な抗腫瘍効果を達成しつつテモゾロミドの使用量を減らすことができ、テモゾロミドに起因する副作用を低減させることができる。 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.
　本発明のテモゾロミド活性増強剤を投与する方法は特に制限されず、用途に応じて選択できる。例えば、経口投与、静脈内投与、留置等の外科的処置等を挙げることができる。テモゾロミドは一般に経口投与されることから、テモゾロミド活性増強剤も経口投与されることが好ましい。テモゾロミド活性増強剤とテモゾロミドとは同時に投与しても、間隔をあけて投与してもよい。本発明のテモゾロミド活性増強剤は、使用態様に応じて特定ペプチド以外の成分を含んでいてもよく、その形態も特に制限されず、用途に応じて選択できる。特定ペプチド以外に含まれてもよい成分及び形態の具体例は、本発明のＰＤＧＦ依存性細胞増殖抑制剤又は細胞分散抑制剤に関して記載した具体例と同様である。 The method for administering the temozolomide activity enhancer 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. Since 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.
＜抗腫瘍剤＞
　本発明の抗腫瘍剤は、テモゾロミドと、以下の（ａ）又は（ｂ）であるペプチド（特定ペプチド）と、を有効成分として含む。
（ａ）Ｔｈｒ－Ｇｌｕ－Ａｌａ－Ｔｈｒ－Ｉｌｅ－Ｔｈｒ－Ｇｌｙ－Ｌｅｕ－Ｇｌｕ－Ｐｒｏ－Ｇｌｙ－Ｔｈｒ－Ｇｌｕ－Ｔｙｒ－Ｔｈｒ－Ｉｌｅ－Ｔｙｒ－Ｖａｌ－Ｉｌｅ－Ａｌａ－Ｌｅｕ（配列番号１）で表されるアミノ酸配列からなるペプチド。
（ｂ）アミノ酸配列（ａ）において１若しくは数個のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなり、かつテモゾロミド活性増強作用を有するペプチド。 <Anti-tumor agent>
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) 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 temozolomide activity enhancing action.
　特定ペプチドが（ｂ）：アミノ酸配列（ａ）において１若しくは数個のアミノ酸が欠失、置換若しくは付加されてなるペプチドである場合、欠失、置換若しくは付加されるアミノ酸残基の数は特定ペプチドがテモゾロミド活性増強作用を有する範囲であれば特に制限されない。例えば、１～９個であり、１～６個であることが好ましく、１～４個であることがより好ましい。合成の効率、取扱性、安定性等の観点からは、（ｂ）のペプチドのアミノ酸残基の総数は３０以下であることが好ましく、２８以下であることがより好ましく、２５以下であることがより好ましい。 When 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. For example, the number is 1 to 9, preferably 1 to 6, and more preferably 1 to 4. From the viewpoint of synthesis efficiency, handleability, stability, etc., 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.
　本発明の抗腫瘍剤は、テモゾロミドと特定ペプチドとを有効成分として含む混合剤であっても、テモゾロミドと特定ペプチドとをそれぞれ有効成分として含む薬剤の組み合わせであってもよい。本発明の抗腫瘍剤を投与する方法は特に制限されず、用途に応じて選択できる。例えば、経口投与、静脈内投与、留置等の外科的処置等を挙げることができる。本発明の抗腫瘍剤が混合剤である場合、テモゾロミドは一般に経口投与されることから、経口投与されることが好ましい。 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. When the antitumor agent of the present invention is a mixed agent, temozolomide is generally administered orally and is preferably administered orally.
　本発明の抗腫瘍剤がテモゾロミドと特定ペプチドとをそれぞれ有効成分として含む薬剤の組み合わせである場合の具体的態様としては、テモゾロミドを含む第一の収容部と、特定ペプチドを含む第二の収容部と、を備えた態様が挙げられる。第一の収容部及び第二の収容部は、それぞれの薬剤を独立して収容できるものであれば特に制限はない。例えば、テモゾロミドを収容する部分と特定ペプチドを収容する部分とを有する容器の形態や、テモゾロミドを収容する部分と特定ペプチドを収容する部分とが区分けされているシートの形態であってもよい。 As a specific embodiment in the case where 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. For example, the form of the container which has the part which accommodates a temozolomide, and the part which accommodates a specific peptide, and the form of the sheet | seat in which the part which accommodates a temozolomide, and the part which accommodates a specific peptide may be sufficient.
＜神経膠腫治療方法＞
　神経膠腫治療方法は、以下の（ａ）又は（ｂ）であるペプチド（特定ペプチド）を患者に投与することを含む。
（ａ）Ｔｈｒ－Ｇｌｕ－Ａｌａ－Ｔｈｒ－Ｉｌｅ－Ｔｈｒ－Ｇｌｙ－Ｌｅｕ－Ｇｌｕ－Ｐｒｏ－Ｇｌｙ－Ｔｈｒ－Ｇｌｕ－Ｔｙｒ－Ｔｈｒ－Ｉｌｅ－Ｔｙｒ－Ｖａｌ－Ｉｌｅ－Ａｌａ－Ｌｅｕ（配列番号１）で表されるアミノ酸配列からなるペプチド。
（ｂ）アミノ酸配列（ａ）において１若しくは数個のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ抗神経膠腫作用を有するペプチド。 <Glioma treatment method>
The glioma treatment method includes administering a peptide (specific peptide) which is the following (a) or (b) to a patient.
(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 an antiglioma action.
　特定ペプチドが（ｂ）：アミノ酸配列（ａ）において１若しくは数個のアミノ酸が欠失、置換若しくは付加されてなるペプチドである場合、欠失、置換若しくは付加されるアミノ酸残基の数は特定ペプチドが抗神経膠腫作用を有する範囲であれば特に制限されない。例えば、１～９個であり、１～６個であることが好ましく、１～４個であることがより好ましい。合成の効率、取扱性、安定性等の観点からは、（ｂ）のペプチドのアミノ酸残基の総数は３０以下であることが好ましく、２８以下であることがより好ましく、２５以下であることがより好ましい。 When 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 | limit in particular. For example, the number is 1 to 9, preferably 1 to 6, and more preferably 1 to 4. From the viewpoint of synthesis efficiency, handleability, stability, etc., 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.
　本発明者らは、上記アミノ酸配列を有する特定ペプチドが神経膠腫の治療に有効であることを見出した。ここで、「治療」には神経膠腫に起因する症状を消失又は軽減させることのほか、症状の進行の度合いを抑制することも含まれる。「抗神経膠腫作用」には、神経膠腫を消失又は縮小させる作用のほか、神経膠腫の増大、移動、浸潤等を抑制する作用も含まれる。特定ペプチドを患者に投与する方法は特に制限されず、経口投与、静脈内投与、外科的処置等を挙げることができる。患者に投与する特定ペプチドの量は特に制限されず、腫瘍の状態、特定ペプチドとともに使用する他の成分の種類や量等に応じて選択できる。本発明の神経膠腫治療方法は、特定ペプチドを有効成分とする薬剤の単独使用であっても、テモゾロミド等のその他の薬剤との併用であってもよい。 The present inventors have found that a specific peptide having the above amino acid sequence is effective for the treatment of glioma. Here, "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.
　以下に実施例を挙げて、本発明をさらに具体的に説明する。以下の実施例に示す材料、使用量、割合、処理手順等は、本発明の趣旨を逸脱しない限り適宜変更することができる。したがって、本発明の範囲は以下に示す具体例により限定的に解釈されるべきものではない。 The present invention will be described more specifically with reference to the following examples. The materials, amounts used, ratios, processing procedures, and the like shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.
＜実施例１　ＰＤＧＦ依存性細胞増殖抑制効果の評価＞
　特定ペプチドによるＰＤＧＦ依存性の細胞増殖の抑制効果を以下の試験によって評価した。
　ヒト神経膠芽腫（グリオブラストーマ）細胞株Ｔ９８Ｇを１０％ＦＢＳを含むＲＰＭＩ１６４０で３７℃、５％ＣＯ２の条件でインキュベータ内で培養した。培地には０．１５％　ＮａＨＣＯ３、２ｍＭ　Ｌ－ｇｌｕｔａｍｉｎｅ、Ｐｅｎｉｃｉｌｌｉｎ－Ｓｔｒｅｐｔｏｍｙｃｉｎ　Ｓｏｌｕｔｉｏｎ（和光純薬工業株式会社）を添加した。ＦＮコート（０．２５μｇ／ｍＬ）した９６ウェルプレートに培養したＴ９８Ｇ細胞を所定量のＰＤＧＦ（和光純薬工業株式会社）、ＴＮＩＩＩＡ２（配列番号２）及びＦＮＩＩＩ１４（配列番号１）とともに播種し、３７℃、５％ＣＯ２の条件でインキュベータ内で培養した。その後、Ｃｅｌｌ　Ｃｏｕｎｔｉｎｇ　Ｋｉｔ－８（株式会社同仁化学研究所）を用いて生存細胞数を測定した。吸光度測定にはＭｕｌｔｉｍｏｄｅ　Ｐｌａｔｅ　Ｒｅａｄｅｒ　ＡＲＶＯＴＭ　Ｘ４（Ｐｅｒｋｉｎ　Ｅｌｍｅｒ社）を用いた。 <Example 1 Evaluation of PDGF-dependent cell proliferation inhibitory effect>
The inhibitory effect of PDGF-dependent cell proliferation by specific peptides was evaluated by the following test.
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 μg / 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 ™ X4 (Perkin Elmer) was used for the absorbance measurement.
　結果を図１に示す。図中の数字はＴＮＩＩＩＡ２の添加量（μｇ／ｍＬ）を示す。「－ＰＤＧＦ」はＰＤＧＦを添加しなかった群を、「＋ＰＤＧＦ」はＰＤＧＦを１０ｎｇ／ｍＬ添加した群を示す。「＋ＦＮＩＩＩ１４　２５」はＦＮＩＩＩ１４を２５μｇ／ｍＬ添加した群を、「＋ＦＮＩＩＩ１４　５０」はＦＮＩＩＩ１４を５０μｇ／ｍＬ添加した群を示す。図中のエラーバーは標準偏差（ｎ＝３）を示す。 The results are shown in FIG. The numbers in the figure indicate the amount of TNIIIA2 added (μg / mL). “−PDGF” represents a group to which PDGF was not added, and “+ 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 μg / mL, and “+ FNIII14 50” represents a group to which FNIII14 was added at 50 μg / mL. Error bars in the figure indicate standard deviation (n = 3).
　図１のグラフに示すように、ＰＤＧＦを添加しない場合に比べてＰＤＧＦを添加した場合ではＴＮＩＩＩＡ２の添加によるＴ９８Ｇ細胞の増加が顕著にみられ、ＴＮＩＩＩＡ２がＰＤＧＦ依存性の細胞増殖を促進することがわかった。さらに、ＦＮＩＩＩ１４を添加した場合はＴ９８Ｇ細胞の増加が顕著に抑制され、ＦＮＩＩＩ１４がＴＮＩＩＩＡ２によるＰＤＧＦ依存性の細胞増殖の促進を抑制することがわかった。 As shown in the graph of FIG. 1, when PDGF is added compared to the case where PDGF is not added, the increase of T98G cells due to the addition of TNIIIA2 is significantly observed, and TNIIIA2 promotes PDGF-dependent cell proliferation. all right. Furthermore, when FNIII14 was added, the increase in T98G cells was remarkably suppressed, and it was found that FNIII14 suppresses the promotion of PDGF-dependent cell proliferation by TNIIIA2.
＜実施例２　足場非依存性細胞増殖抑制効果の評価＞
　特定ペプチドによる足場非依存性細胞増殖の抑制効果を軟寒天コロニーアッセイによって評価した。足場非依存性の細胞増殖能は細胞のがん化の指標であり、増殖が活発であるほど細胞のがん化が著しいことを意味する。
　１．４％アガロースゲル（Ｂａｃｔｏ　Ａｇａｒ、ＢＤ社）をオートクレーブで滅菌し、溶解した後、恒温槽で４５℃にした。同じく４５℃で２０％ＦＢＳを含む２倍濃度の培地をアガロースゲルと等量混合し、１２ウェルプレートに１ｍＬ添加し、室温で固めた。上記で等量混合したアガロースゲルを、試薬及び実施例１に記載の方法で培養したＴ９８Ｇ細胞の懸濁液と更に等量混合し、各ウェルに１ｍＬ添加した。４℃で８分間静置して固めた後、１０％ＦＢＳを含む培地を各ウェルに１ｍＬ加えた。３７℃、５％ＣＯ２の条件でインキュベータで一定期間培養し、その後コロニー数をカウントした。 <Example 2 Evaluation of inhibitory effect on anchorage-independent cell proliferation>
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. Similarly, 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.
　結果を図２に示す。図中の「ｃｏｎｔｒｏｌ」は試薬を添加しなかった群、「ＴＮＩＩＩＡ２」はＴＮＩＩＩＡ２を２５μｇ／ｍＬ添加した群、「ＰＤＧＦ」はＰＤＧＦを１０ｎｇ／ｍＬ添加した群、「ＴＮＩＩＩＡ２＋ＰＤＧＦ」はＴＮＩＩＩＡ２を２５μｇ／ｍＬ及びＰＤＧＦを１０ｎｇ／ｍＬ添加した群を示す。図中のエラーバーは標準偏差（ｎ＝３）を示す。 The results are shown in FIG. In the figure, “control” is a group in which no reagent is added, “TNIIIA2” is a group in which TNIIIA2 is added at 25 μg / mL, “PDGF” is a group in which PDGF is added at 10 ng / mL, and “TNIIIA2 + PDGF” is TNIIIA2 at 25 μg / mL. And the group which added 10 ng / mL of PDGF is shown. Error bars in the figure indicate standard deviation (n = 3).
　図２のグラフ及び写真に示すように、ＴＮＩＩＩＡ２はＴ９８Ｇ細胞の足場非依存性増殖を増強し、その増強効果はＰＤＧＦを添加した場合により顕著であることがわかった。さらに、ＦＮＩＩＩ１４を添加した場合はＴ９８Ｇ細胞の足場非依存性増殖が顕著に抑制され、ＦＮＩＩＩ１４がＴＮＩＩＩＡ２による足場非依存性増殖の促進を抑制することがわかった。 As shown in the graph and photograph of FIG. 2, 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.
＜実施例３　腫瘍細胞分散抑制効果の評価＞
　特定ペプチドによる腫瘍細胞分散の抑制効果をｓｃａｔｔｅｒｉｎｇ　ａｓｓａｙによって評価した。細胞の分散が活発であるほど腫瘍の移動や浸潤が著しいことを意味する。
　ＦＮコート（０．２５μｇ／ｍＬ）したプレートに実施例１に記載の方法で培養したＴ９８Ｇ細胞を播種し、３７℃、５％ＣＯ２の条件でインキュベータで培養した。敷石状に増えた細胞に各試薬を反応させ、一定時間後にＤｉｆｆ－Ｑｕｉｋ（シスメックス株式会社）で固定、染色し、細胞の形態変化を観察した。細胞の形態変化はＭｏｔｉｃ　Ｉｍａｇｅ　Ｐｌｕｓ　２．２Ｓ（株式会社島津理化）で撮影した。 <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 μg / 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.).
　結果を図３に示す。図中の「ｃｏｎｔｒｏｌ」は試薬を添加しなかった群、「ＴＮＩＩＩＡ２」はＴＮＩＩＩＡ２を２５μｇ／ｍＬ添加した群、「ＰＤＧＦ」にはＰＤＧＦを１０ｎｇ／ｍＬ添加した群、「ＴＮＩＩＩＡ２＋ＰＤＧＦ」はＴＮＩＩＩＡ２を２５μｇ／ｍＬ及びＰＤＧＦを１０ｎｇ／ｍＬ添加した群を示す。図中のエラーバーは標準偏差（ｎ＝３）を示す。 The results are shown in FIG. In the figure, “control” is a group to which no reagent was added, “TNIIIA2” was a group to which TNIIIA2 was added at 25 μg / mL, “PDGF” was a group to which PDGF was added at 10 ng / mL, and “TNIIIA2 + PDGF” was TNIIIA2 at 25 μg / mL. The group which added 10 ng / mL of mL and PDGF is shown. Error bars in the figure indicate standard deviation (n = 3).
　図３の左側の写真に示すように、ＴＮＩＩＩＡ２はＴ９８Ｇ細胞の分散を誘導することがわかった。さらに、ＴＮＩＩＩＡ２はＰＤＧＦの添加の有無とは無関係にＴ９８Ｇ細胞の分散を誘導することがわかった。他方、図３の左側の写真に示すようにＦＮＩＩＩ１４を添加した場合はＴＮＩＩＩＡ２を添加した場合でもＴ９８Ｇ細胞の分散がみられず、ＦＮＩＩＩ１４がＴＮＩＩＩＡ２による細胞分散の誘導を阻害することがわかった。 As shown in the photograph on the left side of FIG. 3, it was found that 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.
＜実施例４　マウス側背部における腫瘍形成抑制効果の評価＞
　特定ペプチドによる腫瘍形成の抑制効果をマウスを用いた動物実験によって評価した。６週令雄のヌードマウス（Ｂａｌｂ／ｃ　ｎｕ／ｎｕ）の左背部の皮下にラットグリオーマ細胞株９Ｌ（５×１０５　ｃｅｌｌｓ／１００μＬ）を移植した。腫瘍体積が約５０ｍｍ３になったところでマウスを無作為に二群に分け、一方の群には生体内での分解を防ぐために第２位のグルタミン酸をＤ体に変換したＦＮＩＩＩ１４を、もう一方の群には配列をシャッフルして不活性化したＦＮＩＩＩ１４の変異体（配列番号３）を、個体あたり２５０μｇの量で尾静脈内に連日、２１日間投与した。腫瘍長径及び短径を一日おきにノギスで測定し、腫瘍体積（Ｖ）を腫瘍長径（Ｌ）と腫瘍短径（Ｗ）から、Ｖ＝Ｌ×Ｗ２×０．５の式で算出した。マウスの体重も一日おきに測定した。 <Evaluation of tumor formation inhibitory effect in 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 μL) was transplanted subcutaneously into the left back of a 6-week-old male nude mouse (Balb / c nu / nu). When the tumor volume reached about 50 mm 3 , 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 μg per individual. The tumor major axis and minor axis were measured with a caliper every other day, and the tumor volume (V) was calculated from the tumor major axis (L) and tumor minor axis (W) by the formula V = L × W 2 × 0.5. . Mouse weights were also measured every other day.
　結果を図４に示す。図４中の「ｃｏｎｔｒｏｌ　ｐｅｐｔｉｄｅ」はＦＮＩＩＩ１４の変異体を投与したコントロール群、「Ｄ－Ｇｌｕ　ＦＮＩＩＩ１４」は第２位のグルタミン酸をＤ体に変換したＦＮＩＩＩ１４を投与した群の腫瘍体積（ｍｍ３）及び体重（ｍｇ）の変化をそれぞれ示す。図中のエラーバーは標準偏差（ｎ＝５）を示す。 The results are shown in FIG. In FIG. 4, “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, and Each change in body weight (mg) is shown. Error bars in the figure indicate standard deviation (n = 5).
　図４の左側のグラフに示すように、ＦＮＩＩＩ１４を投与した群はコントロール群に比べて腫瘍体積の増大が顕著に抑制されており、ＦＮＩＩＩ１４が腫瘍の形成を抑制することがわかった。また、右側のグラフに示すように、ＦＮＩＩＩ１４を投与した群の体重の変化はコントロール群に比べて大きく変わらなかった。 As shown in the graph on the left side of FIG. 4, in the group administered with FNIII14, the increase in tumor volume was significantly suppressed as compared with the control group, and it was found that FNIII14 suppressed tumor formation. Further, as shown in the graph on the right side, the change in body weight of the group administered with FNIII14 was not significantly different from that in the control group.
＜実施例５　テモゾロミド活性増強効果の評価＞
　特定ペプチドによるテモゾロミド活性の増強効果を以下の試験によって評価した。
　ＦＮコート（０．２５μｇ／ｍＬ）したプレートに実施例１に記載の方法で培養したＴ９８Ｇ細胞を播種し、３７℃、５％ＣＯ２の条件でインキュベータで培養した。培養した細胞に対し、テモゾロミド単独又はテモゾロミドとＦＮＩＩＩ１４との組みあわせを濃度を変化させて作用させ、細胞の生存率を調べた。 <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 μg / 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.
　結果を図５に示す。図５のグラフ中の「Ｔｅｍｏｚｏｌｏｍｉｄｅ」はテモゾロミド単独を作用させた群、「ＴＮＩＩＩ１４＋Ｔｅｍｏｚｏｌｏｍｉｄｅ」はテモゾロミドとＦＮＩＩＩ１４との組み合わせを作用させた群の細胞生存率の変化を示す。図中のエラーバーは標準偏差（ｎ＝３）を示す。 The results are shown in FIG. In the graph of FIG. 5, “Temozolomide” indicates a change in cell viability of a group in which temozolomide alone was allowed to act, and “TNIII14 + Temozolomide” indicates a change in cell survival rate in a group to which a combination of temozolomide and FNIII14 was allowed to act. Error bars in the figure indicate standard deviation (n = 3).
　テモゾロミドとＦＮＩＩＩ１４の併用効果について既報（Ｄｒｅｗｉｎｋｏら、Ｃａｎｃｅｒ　Ｔｒｅａｔ　Ｒｅｐ　６０（１１）１６１９－１６２５（１９７６））に従い、下記の式によってＣｏｍｂｉｎａｔｉｏｎ　Ｉｎｄｅｘ（ＣＩ）を算出した。ＣＩの値が１より小さいことは、テモゾロミドとＦＮＩＩＩ１４の併用により相乗効果が現れていることを意味する。
　ＣＩ＝Ｖ３／（Ｖ１×Ｖ２）　
　Ｖ１；抗がん剤（テモゾロミド）をある濃度で作用させた時の細胞生存率
　Ｖ２；被験物質（ＦＮＩＩＩ１４）をある濃度で作用させた時の細胞生存率
　Ｖ３；両者を併用して作用させた時の細胞生存率
　ＣＩ＞１：拮抗効果、ＣＩ＝１：相加効果、ＣＩ＜１：相乗効果 According to the previous report (Drewinko et al., Cancer Treat Rep 60 (11) 1619-1625 (1976)) on the combined effect of temozolomide and FNIII14, 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.
CI = V 3 / (V 1 × V 2 )
V 1 ; cell viability when an anticancer drug (temozolomide) is allowed to act at a certain concentration V 2 ; cell viability when a test substance (FNIII14) is caused to act at a certain concentration V 3 ; Cell survival rate upon action CI> 1: antagonistic effect, CI = 1: additive effect, CI <1: synergistic effect
　図５に示すように、テモゾロミドとＦＮＩＩＩ１４との組み合わせを作用させた細胞の生存率はテモゾロミド単独を作用させた群よりも低く、ＦＮＩＩＩ１４がテモゾロミドの活性を増強することがわかった。さらに、テモゾロミドとＦＮＩＩＩ１４とを併用した場合のＣＩの値は１よりも小さく、相乗効果が現れていることがわかった。 As shown in FIG. 5, 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.
＜使用した試薬及び細胞＞
　ＦＮＩＩＩ１４（ＴＥＡＴＩＴＧＬＥＰＧＴＥＹＴＩＹＶＩＡＬ、配列番号１）、ＴＮＩＩＩＡ２（ＲＳＴＤＬＰＧＬＫＡＡＴＨＹＴＩＴＩＲＧＶＣ、配列番号２）、ＦＮＩＩＩ１４変異体（ＴＥＡＴＩＴＧＬＥＰＧＴＥＹＴＡＹＶＡＡＬＣ、配列番号３）はそれぞれオペロンバイオテクノロジー株式会社より購入した。Ｄ－Ｇｌｕ　ＦＮＩＩＩ１４（Ｔ－［Ｄ－Ｇｌｕ］－ＡＴＩＴＧＬＥＰＧＴＥＹＴＩＹＶＩＡＬ）は佐賀大学大学院工学系研究科の研究グループとの共同研究によって調製した。ヒト神経膠芽腫細胞株Ｔ９８ＧはＡｍｅｒｉｃａｎ　Ｔｙｐｅ　Ｃｕｌｔｕｒｅ　Ｃｏｌｌｅｃｔｉｏｎより購入した。ラットグリオーマ細胞株９Ｌは筑波大学医学医療系脳神経外科学教室より提供を受けた。 <Reagents and cells used>
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) was prepared in collaboration with a research group at the Graduate School of Engineering, Saga University. Human glioblastoma cell line T98G was purchased from American Type Culture Collection. Rat glioma cell line 9L was provided by the Department of Neurosurgery, School of Medicine, University of Tsukuba.

Claims (10)

  1. 　以下の（ａ）又は（ｂ）であるペプチドを有効成分として含む、ＰＤＧＦ依存性細胞増殖抑制剤。
    （ａ）Ｔｈｒ－Ｇｌｕ－Ａｌａ－Ｔｈｒ－Ｉｌｅ－Ｔｈｒ－Ｇｌｙ－Ｌｅｕ－Ｇｌｕ－Ｐｒｏ－Ｇｌｙ－Ｔｈｒ－Ｇｌｕ－Ｔｙｒ－Ｔｈｒ－Ｉｌｅ－Ｔｙｒ－Ｖａｌ－Ｉｌｅ－Ａｌａ－Ｌｅｕ（配列番号１）で表されるアミノ酸配列からなるペプチド。
    （ｂ）アミノ酸配列（ａ）において１若しくは数個のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなり、ＰＤＧＦ依存性細胞増殖抑制作用を有するペプチド。     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.
    (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.
  2. 　前記細胞はＰＤＧＦ受容体を発現する、肉腫及び癌腫からなる群より選択される少なくとも１種の細胞である請求項１に記載のＰＤＧＦ依存性細胞増殖抑制剤。 The PDGF-dependent cell growth inhibitor according to claim 1, wherein the cell is at least one cell selected from the group consisting of sarcoma and carcinoma that expresses a PDGF receptor.
  3. 　前記細胞は神経膠腫、繊維肉腫、骨肉腫、乳がん及び肺がんからなる群より選択される少なくとも１種の細胞である、請求項１又は請求項２に記載のＰＤＧＦ依存性細胞増殖抑制剤。 The PDGF-dependent cell growth inhibitor according to claim 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.
  4. 　請求項１～請求項３のいずれか１項に記載のＰＤＧＦ依存性細胞増殖抑制剤を細胞に接触させることを含む、ＰＤＧＦ依存性細胞増殖の抑制方法。 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 claims 1 to 3.
  5. 　以下の（ａ）又は（ｂ）であるペプチドを有効成分として含む、細胞分散抑制剤。
    （ａ）Ｔｈｒ－Ｇｌｕ－Ａｌａ－Ｔｈｒ－Ｉｌｅ－Ｔｈｒ－Ｇｌｙ－Ｌｅｕ－Ｇｌｕ－Ｐｒｏ－Ｇｌｙ－Ｔｈｒ－Ｇｌｕ－Ｔｙｒ－Ｔｈｒ－Ｉｌｅ－Ｔｙｒ－Ｖａｌ－Ｉｌｅ－Ａｌａ－Ｌｅｕ（配列番号１）で表されるアミノ酸配列からなるペプチド。
    （ｂ）アミノ酸配列（ａ）において１若しくは数個のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなり、細胞分散抑制作用を有するペプチド。     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. 　前記細胞は細胞間接着によって形成された集団又は集隗からの細胞の解離が生じる、肉腫及び癌腫からなる群より選択される少なくとも１種の細胞である請求項５に記載の細胞分散抑制剤。 The cell dispersion inhibitor according to claim 5, wherein the cell is at least one cell selected from the group consisting of a sarcoma and a carcinoma in which dissociation of cells from a population formed by cell-cell adhesion or from a congregation occurs.
  7. 　前記細胞は神経膠腫、繊維肉腫、骨肉腫、乳がん及び肺がんからなる群より選択される少なくとも１種の細胞である、請求項５又は請求項６に記載の細胞分散抑制剤。 The cell dispersion inhibitor according to claim 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. 　請求項５～請求項７のいずれか１項に記載の細胞分散抑制剤を細胞に接触させることを含む、細胞分散の抑制方法。 A method for inhibiting cell dispersion, comprising bringing the cell dispersion inhibitor according to any one of claims 5 to 7 into contact with cells.
  9. 　以下の（ａ）又は（ｂ）であるペプチドを有効成分として含む、テモゾロミド活性増強剤。
    （ａ）Ｔｈｒ－Ｇｌｕ－Ａｌａ－Ｔｈｒ－Ｉｌｅ－Ｔｈｒ－Ｇｌｙ－Ｌｅｕ－Ｇｌｕ－Ｐｒｏ－Ｇｌｙ－Ｔｈｒ－Ｇｌｕ－Ｔｙｒ－Ｔｈｒ－Ｉｌｅ－Ｔｙｒ－Ｖａｌ－Ｉｌｅ－Ａｌａ－Ｌｅｕ（配列番号１）で表されるアミノ酸配列からなるペプチド。
    （ｂ）アミノ酸配列（ａ）において１若しくは数個のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなり、テモゾロミド活性増強作用を有するペプチド。     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).
  10. 　テモゾロミドと、以下の（ａ）又は（ｂ）であるペプチドと、を有効成分として含む抗腫瘍剤。
    （ａ）Ｔｈｒ－Ｇｌｕ－Ａｌａ－Ｔｈｒ－Ｉｌｅ－Ｔｈｒ－Ｇｌｙ－Ｌｅｕ－Ｇｌｕ－Ｐｒｏ－Ｇｌｙ－Ｔｈｒ－Ｇｌｕ－Ｔｙｒ－Ｔｈｒ－Ｉｌｅ－Ｔｙｒ－Ｖａｌ－Ｉｌｅ－Ａｌａ－Ｌｅｕ（配列番号１）で表されるアミノ酸配列からなるペプチド。
    （ｂ）アミノ酸配列（ａ）において１若しくは数個のアミノ酸残基が欠失、置換若しくは付加されたアミノ酸配列からなり、テモゾロミド活性増強作用を有するペプチド。     An antitumor agent comprising temozolomide and the following 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) 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).
PCT/JP2015/067651 2014-06-18 2015-06-18 Pdgf-dependent cell-growth inhibitor, pdgf-dependent cell-growth inhibiting method, cell dispersion inhibitor, cell dispersion inhibiting method, temozolomide activity enhancer, and antitumor agent WO2015194643A1 (en)

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