JP2017178943A - Anti-angiogenetic agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a medicine that inhibits tumor angiogenesis derived from a tumor stem cell, and a therapeutic agent against VEGF inhibitor resistive cancer.SOLUTION: There is provided an inhibitor to angiogenesis derived from a tumor stem cell which contains an antipsychotic agent, particularly at least one antipsychotic agent selected from a selective serotonin reuptake inhibitor (SSRI) or a tricyclic antidepressant, in particular, the inhibitor to angiogenesis derived from a tumor stem cell for treating VEGF pathway inhibitor resistive cancer, and a cancer therapeutic agent in which the inhibitor to angiogenesis derived from a tumor stem cell and the VEGF pathway inhibitor are combined.SELECTED DRAWING: None

Description

  The present invention relates to a tumor stem cell-derived angiogenesis inhibitor, and more particularly to a drug that inhibits angiogenesis by brain tumor stem cells.

  Molecularly targeted drugs for malignant tumors, particularly cancer, are currently being actively researched and developed. Unlike conventional drugs called classical anticancer drugs, molecular targeted drugs act on cancer-specific molecules and have various side effects that are problematic with conventional anticancer drugs. It is a very low drug. However, the research requires enormous costs and a lot of effort over a very long period, from the identification of the target molecule to the screening of various small molecule drugs. In addition, even if such molecularly targeted drugs come to the world through extremely high costs and labor, it is often seen that they cause unexpected complications and do not bring about the effects expected in the experiment.

  The concept of anti-angiogenic therapy for cancer has long been proposed since the 1970s, and it is a treatment that makes cancer more aggressive by inhibiting tumor angiogenesis, which is abnormally increasing for tumors. . As angiogenesis inhibitors as molecular targeting drugs, bevacizumab, sorafenib, sunitinib, pazopanib, vantedanib, aflibercept, and axitinib have been approved, and all these angiogenesis inhibitors are vascular endothelial growth factor (VEGF) VEGF-targeted therapeutic agents that target the signal transduction pathway. Among them, bevacizumab and axitinib are used clinically. Bevacizumab is an anti-human VEGF monoclonal antibody that binds specifically to human VEGF, thereby blocking the binding of VEGF to the VEGF receptor expressed on vascular endothelial cells and the biological activity of VEGF in tumor tissues. Suppresses angiogenesis and inhibits tumor growth. Axitinib inhibits phosphorylation of the vascular endothelial growth factor receptors (VEGFR-1, VEGFR-2 and VEGFR-3) and inhibits downstream signaling. In recent years, bevacizumab has established a strong position as a standard treatment for unresectable advanced / recurrent colorectal cancer in Japan and overseas, but its spread to other cancers has not progressed slowly. The following two reports have been reported in clinical trials for typical malignant brain tumors (glioblastoma) using anti-angiogenic drugs.

The first is AVAglio, an international joint research for malignant brain tumors (glioblastoma) centered in North America. The standard treatment (surgical treatment + alkylating agent + radiotherapy) is the anti-angiogenic drug bevacizumab (Avastin (registered) (Trademark))) is a clinical study with a combination treatment. During treatment with 60 Gy (2 Gy / day × 30 times), temozolomide was administered daily (75 mg / m ² / day), and the anti-angiogenic drug bevacizumab was administered (10 mg / kg every 2 weeks). . Thereafter, administration of bevacizumab was continued for 12 cycles at intervals of 3 weeks. 637 patients were treated, and the median overall survival was 15.7 months in the bevacizumab group and 16.1 months in the non-use group. No significant difference was observed, and conversely, side effects were observed in the bevacizumab group. It has been reported.

  The second is a clinical trial similar to the above-mentioned Abaglio conducted in the international joint research RTOG 0825 for malignant brain tumors (glioblastoma) mainly in Europe. The 2-year survival rate was 33.9% in the bevacizumab (Avastin (registered trademark)) group and 30.1% in the non-use group, and there were many adverse events in the bevacizumab group. Has been reported.

  Furthermore, in the application of a VEGF inhibitor, no improvement in the survival rate has been observed for metastatic breast cancer and primary breast cancer.

  In glioblastoma, which is one of the cancers that have not been treated with VEGF inhibitors such as bevacizumab, tumor stem cells differentiate into endothelial cells, and angiogenesis is possible without depending on VEGF. It has been reported (Non-Patent Document 1).

  On the other hand, the present inventors have found that fluvoxamine or a derivative thereof has an effect of suppressing invasion of cancer cells in the normal brain as a drug that can cross the blood-brain barrier (BBB), and is disclosed in Patent Document 1. . Similar to this fluvoxamine, antipsychotics such as selective serotonin reuptake inhibitors (SSRIs), tricyclic antidepressants, and tetracyclic antidepressants currently in clinical use act in the normal brain. It passes the blood-brain barrier and exhibits high brain migration. Fluvoxamine, which is one of SSRIs, is mainly used as a therapeutic agent for depression, anxiety disorder, obsessive compulsive disorder, eating disorder, attention deficit / hyperactivity disorder, premenstrual syndrome and the like. Fluvoxamine maleate tablets that received the earliest approval of SSRIs in Japan are sold as “Depromer (registered trademark)” (Meiji Seika Pharma Co., Ltd.) and “Lubox (registered trademark)” (Abbvy GK) Has been.

JP 2014-108930 A

Soda Y, et al, Proc. Natl. Acad. Sci., 108 (11): 4274-4280, 2011.

  As described above, existing angiogenesis inhibitors were effective in some cancers such as colorectal cancer, but were ineffective in many malignant tumors including malignant brain tumors. Patent Document 1 suggests that one of the causes is VEGF-independent angiogenesis by tumor stem cells present in the tumor. Non-Patent Document 1 specifically describes a VEGF inhibitor-resistant cancer. There is no indication for the treatment.

  In addition, in Patent Document 1, although there is a slight suggestion of inhibition of normal tumor angiogenesis, specifically, fluvoxamine or a derivative thereof only discloses a cell infiltration suppressing action.

  Therefore, an object of the present invention is to provide a drug that inhibits tumor angiogenesis derived from tumor stem cells and to provide a therapeutic drug for VEGF pathway inhibitor resistant cancer.

  In the normal tumor, the present inventors occupy 80 to 90% of tumor blood vessels composed of growth tissues, and the remaining 10 to 20% are tumor blood vessels derived from tumor stem cells. By suppressing the growth of blood vessels, tumor blood vessels derived from tumor stem cells proliferate in order to supplement the blood vessels, and the proportion increases, and resistance to anti-VEGF treatment is found. By testing various antipsychotic drugs, For the first time, a drug that inhibits tumor blood vessels derived from brain tumor stem cells was discovered and the present invention was completed.

That is, the present invention
[1] Tumor stem cell-derived angiogenesis inhibitors including antipsychotics,
[2] The tumor stem cell-derived angiogenesis inhibitor according to [1] above, wherein the antipsychotic is at least one selected from a selective serotonin reuptake inhibitor (SSRI) or a tricyclic antidepressant;
[3] The tumor stem cell-derived angiogenesis inhibitor according to the above [1] or [2], wherein the antipsychotic is SSRI,
[4] The tumor stem cell-derived angiogenesis inhibitor according to [2] above, wherein the antipsychotic is at least one selected from the group consisting of fluvoxamine, sertraline, paroxetine and imipramine,
[5] The tumor stem cell-derived angiogenesis inhibitor according to any one of the above [1] to [4], which is for treatment of VEGF pathway inhibitor resistant cancer,
[6] A cancer therapeutic agent comprising a combination of the tumor stem cell-derived angiogenesis inhibitor and the VEGF pathway inhibitor according to any one of [1] to [5] above,
[7] The cancer therapeutic agent according to the above [6], which is for treatment of VEGF pathway inhibitor resistant cancer.

  According to the present invention, an effective angiogenesis inhibitor can be provided even for VEGF inhibitor resistant cancer. In addition, since it is an antipsychotic drug whose action on the brain has been confirmed, it can pass through the BBB, and when it is clinically applied as an antipsychotic drug, its side effects are obvious, so Save time and money.

It is a figure of the fluorescence micrograph in the tube formation assay by 005 cell. It is a figure of the fluorescence micrograph which shows the tube formation inhibition ability of fluvoxamine in a tube formation assay. It is a graph which shows the tube formation ability (tube total length) of the control which has not added the chemical | medical agent as 100 with respect to tube formation inhibition by a fluvoxamine. It is a figure of the fluorescence micrograph which shows the tube formation inhibition ability of sertraline in a tube formation assay. It is a graph which shows the tube formation ability (tube full length) of the control which has not added the chemical | medical agent as tube formation inhibition by sertraline as 100. FIG. It is a figure of the fluorescence micrograph which shows the tube formation inhibitory ability of paroxetine in a tube formation assay. It is a graph which shows the tube formation ability (tube full length) of the control which has not added the chemical | medical agent as 100 for tube formation inhibition by paroxetine. It is a figure of the fluorescence micrograph which shows the tube formation inhibition ability of the imipramine in a tube formation assay. It is a graph which shows the tube formation ability (tube full length) of control which has not added the chemical | medical agent as tube formation inhibition by imipramine as 100. FIG. It is a figure of the fluorescence micrograph which shows the tube formation inhibition ability of axitinib in the tube formation assay of a brain tumor stem cell. It is a graph which shows the influence of axitinib on tube formation of a brain tumor stem cell as the tube formation ability (tube total length) of a control without adding a drug as 100. It is a figure of the fluorescence micrograph which shows the tube formation inhibition ability of axitinib in the tube formation assay of a normal brain tumor cell. It is a graph which shows the influence of axitinib on tube formation of normal brain tumor stem cells, with the tube formation ability (tube total length) of a control without adding a drug being 100. It is a figure of the MRI image which shows the effect of the chemical | medical agent in a brain tumor model mouse. (A) is a T2-enhanced image after 2 weeks of treatment, and (b) is a T2-Gd enhanced image after administration of a gadolinium contrast agent after imaging of (a). It is a graph which shows the tumor volume computed from the MRI image of FIG.14 (b). It is a graph which shows the expression of mRNA by RT-PCR in the vascular endothelial cell derived from a tumor stem cell. (A) is a graph comparing the expression levels of five target genes at 6 hours after administration of fluvoxamine, and (b) is Ang1 at 6 hours and 20 hours after administration of fluvoxamine relative to the expression levels of Ang1 before administration of fluvoxamine. It is a graph which shows an expression level relatively. It is a figure of the electrophoresis photograph which shows the expression level of VEGFR2 and TIE2 in each cell.

  The tumor stem cell-derived angiogenesis inhibitor of the present invention is characterized by containing an antipsychotic drug as an active ingredient.

  “Tumor stem cells” are stem cells that are present in a very small percentage of tumor tissue, and are characterized by self-renewal, tumorigenicity, treatment resistance, multipotency, and viability in the microenvironment. Etc. There are three main cancer treatments: surgery, radiation therapy, and chemotherapy, and multidisciplinary treatments combining these techniques are performed depending on the level of the disease. Among them, stereotactic radiotherapy such as Cyberknife has made it possible to control the radiation field in millimeters with the development of medical equipment. However, taking malignant brain tumors as an example, the highest malignant glioblastoma has an average survival time of about 1 year and a 5-year survival rate of less than 5%, despite the development of radiation therapy techniques as described above. Prognosis is poor. The reason for this is that the tumor grows invasively into the brain. A tumor having such a high invasion ability has many tumor cells scattered at the cellular level in the normal brain outside the tumor area depicted on an image such as MRI, and these cells have not been treated. These untreatable cells include tumor stem cells, whose features described above make the prognosis after cancer treatment poor. Among these features, focusing on pluripotency, it has been reported that tumor stem cells differentiate into pericytes and endothelial cells that make up tumor blood vessels, resulting in angiogenesis.

  Angiogenesis derived from tumor stem cells is not inhibited by currently used anti-angiogenic drugs, i.e., anti-angiogenic drugs that target the VEGF pathway, and is born in a proliferation system unrelated to the VEGF pathway. And this angiogenesis derived from tumor stem cells is considered to increase the proliferation instead when the angiogenesis depending on the VEGF pathway generated from the normal growth tissue is inhibited.

  As antipsychotic drugs, pharmacokinetics and side effects have been clarified, and those that have been clinically applied are preferably used. When used for brain tumors, the drug acts in the brain and exhibits high brain migration It is preferable. Such antipsychotics include selective serotonin reuptake inhibitors (SSRI), typical antipsychotics, atypical antipsychotics, benzodiazepines (BZD), BZD receptor antagonists, tricyclic antidepressants, four Examples include cyclic antidepressants. From the viewpoint of its effect, SSRI or tricyclic antidepressant is preferable.

  Specifically, examples of SSRIs include paroxetine, fluoxetine, fluvoxamine, sertraline, (S) -citalopram, citalopram and the like.

  Typical antipsychotics include chlorpromazine.

  Examples of atypical antipsychotics include olanzapine and quetiapine.

  Examples of benzodiazepines include chlordiazepoxide and diazepam.

  BZD receptor antagonists include alprazolam, bromazepam, brotizolam, chlordiazepoxide, clonazepam, chlorazepate, clothiazepam, cloxazolam, diazepam, estazolam, etizolam, ethyl loflazepate, flunitrazepam, flurazepamaze, flurazepamaze, Nitrazepam, prazepam, quazepam, triazolam, clobazam, flumazenil, eszopiclone, zaleplon, zolpidem, zopiclone, and the like.

  Tricyclic antidepressants include imipramine, clomipramine, trimipramine and the like.

  Examples of tetracyclic antidepressants include maprotiline, mianserin, cetiptiline, mirtazapine and the like.

  Among these, from the viewpoint of reducing the time and cost required for clinical application, chlorpromazine, olanzapine, quetiapine, imipramine, clomipramine, trimipramine, paroxetine, fluoxetine, which are currently used as antipsychotics, Fluvoxamine, sertraline, (S) -citalopram, citalopram and the like can be preferably used, and in view of high effects, fluvoxamine, sertraline, paroxetine and imipramine are more preferable, and fluvoxamine is most preferable.

  In addition, the tumor stem cell-derived angiogenesis inhibitor of the present invention can be used in combination with other anticancer agents. As a combination method, any method such as simultaneous administration, sequential administration, and individual administration may be used. Such drugs that can be used in combination are not particularly limited. For example, known anticancer agents having an effect of killing cancer cells, and known anticancer agents having an effect of suppressing tumor growth and metastasis. Examples include cancer drugs.

  The tumor stem cell-derived angiogenesis inhibitor of the present invention is preferably a cancer therapeutic agent combined with a VEGF pathway inhibitor. This suppresses angiogenesis that depends on the VEGF pathway composed of the growth tissue, and prevents an increase in angiogenesis derived from brain tumor stem cells instead of angiogenesis that depends on the VEGF pathway. Compared with the case, it can have a special synergistic effect. As a combination method, any method such as simultaneous administration, sequential administration, and individual administration may be used. Such an anti-angiogenic agent targeting the VEGF pathway is not particularly limited as long as it targets the VEGF pathway, and the first generation of VEGFR antibody (bevacizumab etc.), VEGF receptor inhibitor Examples include sorafenib, sunitinib, vazovanib, everolimus, etc. It can also be used together. In particular, axitinib is more preferable because the combined effect has been confirmed.

  Furthermore, a synergistic effect can be expected also in combination with an integrin inhibitor syringetide (cRGD peptide) having an anti-angiogenic effect, which is preferable.

  The tumor stem cell-derived angiogenesis inhibitor of the present invention and the cancer therapeutic agent of the present invention may be administered as an active ingredient antipsychotic agent or VEGF pathway inhibitor itself, but generally at least 1 It is preferably administered in the form of a pharmaceutical composition comprising one pharmaceutically acceptable excipient.

  The dosage form and administration form of the tumor stem cell-derived angiogenesis inhibitor of the present invention and the cancer therapeutic agent of the present invention are not particularly limited, and examples thereof include tablets, capsules, granules, powders, and syrups. Suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections and the like. These preparations can be prepared according to a conventional method. The liquid preparation may be dissolved or suspended in water or other appropriate solvent at the time of use. Tablets and granules may be coated by a well-known method. In the case of injection, it is prepared by dissolving the compound of the present invention in water, but it may be dissolved in physiological saline or glucose solution as necessary, and a buffer or preservative may be added. Good. Provided in any formulation for oral or parenteral administration. For example, pharmaceutical compositions for oral administration in the form of granules, fine granules, powders, hard capsules, soft capsules, syrups, emulsions, suspensions or liquids, for intravenous administration, for intramuscular administration, Alternatively, it can be prepared as a pharmaceutical composition for parenteral administration in the form of injections, drops, transdermal absorbents, transmucosal absorbents, nasal drops, inhalants, suppositories, etc. for subcutaneous administration. Injections, infusions, and the like can be prepared as powder dosage forms such as lyophilized forms, and can be used after being dissolved in an appropriate aqueous medium such as physiological saline at the time of use. It is also possible to administer a sustained release preparation coated with a polymer directly into the brain.

  The type of pharmaceutical additive used in the production of the pharmaceutical composition of the present invention, the ratio of the pharmaceutical additive to the active ingredient, or the method for producing the pharmaceutical composition is appropriately selected by those skilled in the art according to the form of the composition. It is possible. As the additive for preparation, an inorganic or organic substance, or a solid or liquid substance can be used, and generally it can be blended between 1% by mass and 90% by mass with respect to the active ingredient mass. . Specific examples of such substances include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminate metasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, carboxymethylcellulose calcium, ions Exchange resin, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, bee gum, titanium oxide, sorbitan fatty acid ester, lauryl sulfate Sodium, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polysol Over DOO, macrogol, vegetable oils, waxes, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, water and the like.

  In order to produce solid preparations for oral administration, active ingredients and excipient components such as lactose, starch, crystalline cellulose, calcium lactate, silicic acid, etc. are mixed to form a powder, or as required. Binders such as sucrose, hydroxypropylcellulose, polyvinylpyrrolidone, disintegrants such as carboxymethylcellulose and carboxymethylcellulose calcium are added and wet or dry granulated to form granules. In order to produce tablets, these powders and granules may be tableted as they are or with the addition of lubricants such as magnesium stearate and talc. These granules or tablets are coated with an enteric solvent base such as hydroxypropylmethylcellulose phthalate or methacrylic acid-methyl methacrylate polymer to form an intestinal solvent formulation, or coated with ethylcellulose, carnauba wax, hardened oil, etc. It can also be a formulation. In order to produce capsules, powder capsules or granules are filled into hard capsules, or active ingredients are coated as they are or dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, etc., and then coated with a gelatin film to form soft capsules. be able to.

  In order to produce injections, active ingredients such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc. Dissolve in distilled water for injection together with an isotonic agent, filter aseptically and fill ampoules, or add mannitol, dextrin, cyclodextrin, gelatin, etc. Good. In addition, lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil, etc. may be added to the active ingredient and emulsified in water to give an emulsion for injection.

  The dose and frequency of administration of the tumor stem cell-derived angiogenesis inhibitor of the present invention and the cancer therapeutic agent of the present invention are not particularly limited, and prevention and / or progression of the disease to be treated and / or the purpose of treatment, the type of disease, It is possible to make an appropriate selection according to the judgment of the doctor according to conditions such as the weight and age of the patient and the severity of the disease. In general, the dose per day for an oral administration or injection administration is preferably 0.1 mg or more from the viewpoint of obtaining a sufficient effect as the active ingredient mass of a tumor stem cell-derived angiogenesis inhibitor. 1 mg or more is more preferable, 10 mg or more is further preferable, 25 mg or more is particularly preferable, and 150 mg or more is most preferable. In addition, the dose per day for oral administration is preferably 3000 mg or less, more preferably 300 mg or less, and more preferably 150 mg or less, considering the effects of side effects, etc., as the active ingredient mass of the tumor stem cell-derived angiogenesis inhibitor. More preferred is 100 mg or less, most preferred is 50 mg or less. Each of the tumor stem cell-derived angiogenesis inhibitor of the present invention and the cancer therapeutic agent of the present invention is divided into once or several times a day, or every several days, and in the case of injection, it can be administered continuously. It can be administered by intermittent administration.

  From the viewpoint of obtaining a sufficient effect, the therapeutic agent for cancer of the present invention is 4 mg per day from the viewpoint of obtaining a sufficient effect by using the brain tumor stem cell-derived angiogenesis inhibitor at the above-mentioned dose and the VEGF pathway inhibitor as the active ingredient mass of axitinib. (2 mg once, twice a day) or more is preferable, 6 mg (3 mg once, twice a day) or more is more preferable, 10 mg (5 mg once, twice a day) or more is more preferable, 14 mg (7 mg once, twice daily) or more is particularly preferable. In addition, the dose per day for oral administration is preferably 14 mg or less, more preferably 10 mg or less, and more preferably 6 mg or less, considering the effects of side effects, etc., as the active ingredient mass of the tumor stem cell-derived angiogenesis inhibitor. 4 mg or less is most preferable.

  The tumor stem cell-derived angiogenesis inhibitor of the present invention and the cancer therapeutic agent of the present invention are prevented from being immediately removed from the body as sustained release preparations such as delivery systems encapsulated in implantable tablets and microcapsules. Can be prepared using the resulting carrier. As carriers, biodegradable, biocompatible polymers can be used, such as, for example, ethylene vinyl acetate copolymers, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such materials can be readily prepared by those skilled in the art. Liposome suspensions can also be used as pharmaceutically acceptable carriers. Useful liposomes are prepared as a lipid composition comprising, but not limited to, phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanol (PEG-PE), through a filter of appropriate pore size so as to be of a size suitable for use, and in reverse phase. Purified by evaporation.

  The target of application of the tumor stem cell-derived angiogenesis inhibitor of the present invention and the cancer therapeutic agent of the present invention is any cancer that is desired to inhibit tumor stem cell-derived angiogenesis, and the prognosis of the cancer. For VEGF pathway inhibitor resistant cancer, it is known that tumor stem cell-derived angiogenesis is promoted after VEGF pathway inhibition treatment. Therefore, the tumor stem cell-derived angiogenesis inhibitor of the present invention, It is preferable to apply an anticancer drug.

  A VEGF pathway inhibitor resistant cancer is a state in which resistance to a VEGF pathway inhibitor has been acquired. For example, even if VEGF pathway inhibitor treatment is performed, no therapeutic effect is observed, It means a state in which progression cannot be suppressed, that is, “cancer” in which the prognostic extension effect is not observed. For example, malignant brain tumors (glioblastomas) have not been shown to be prognostic in patients with brain tumors, even if Avastin, a VEGF pathway inhibitor, is administered according to the Avaglio study. Classified as pathway inhibitor resistant cancer.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

First, the cells used in the following Reference Examples, Examples and Comparative Examples and their culture will be described.
1. Cell 1-1. Brain tumor stem cells 005 (hereinafter referred to as 005 cells) were distributed by Dr. Inder M. Verma (Laboratory of Genetics, Salk Institute for Biological Studies, USA). This cell is prepared by the Cre-loxP gene recombination technique as described in Marumoto T. et al., Development of a novel mouse glioma model using lentiviral vectors. Nat. Med. 2009; 15 (1): 110-116. Using a lentivirus, two tumor genes, H-Ras, Akt, and GFP as a reporter gene were introduced to cause a mouse to form a brain tumor and isolated therefrom.

1-2. Human Umbilical Vein Endothelial Cell (HUVEC) was purchased from Cell Applications, USA.

2. Cell culture 2-1.005 cells 005 cells are N ₂ supplement (Thermo Fisher Scientific, Waltham, MA, USA), 100 U / ml penicillin-100 μg / mL streptomycin (Wako Pure Chemical Industries, Ltd.), basic Dulbecco's Modified Eagle containing FGF (Peprotech, Rocky Hill, NJ, USA), Epidermal Growth Factor (EGF) (Prospec, East Brunswick, NJ, USA), Sodium Heparinate (Sigma-Aldrich, St. Louis, MO, USA) An ultra-low adhesion surface culture dish having a diameter of 10 cm using a medium in which a medium (DMEM-low glucose, Thermo Fisher Scientific) and Ham F-12 medium (Wako Pure Chemical Industries, Ltd.) are mixed at a ratio of 1: 1. (Ultra-low attachment culture dishes) (Corning , NY, USA) at 37 ° C. under 21% O ₂ .

2-2. HUVEC
The culture was carried out using 2% FBS, 0.4% hydrocortisone, 0.4% hFGF, 0.1% VEGF, 0.1% R3-IGF, 0.1% ascorbic acid, 0.1% hEGF,. The test was performed using EGM (registered trademark) -2 BulletKit (registered trademark) (Lonza, Switzerland) supplemented with 1% GA-1000 and 0.1% heparin at 37 ° C. and 21% O ₂ .

Reference Example 1: Confirmation that 005 cells are stem cells The expression of CD133, which is a stem cell marker, was confirmed for 005 cells using an antibody under the following conditions.

Dulbecco's modified Eagle medium containing 005 cells in 10% (v / v) fetal bovine serum (GE Healthcare, Fairfield, CA, USA), 100 U / mL penicillin-100 μg / mL streptomycin (Wako Pure Chemical Industries, Ltd.) (DMEM, Wako Pure Chemical Industries, Ltd.) and 12 mm cover glass (AGC Techno Glass Co., Ltd.) coated with poly-L-lysine and suspended in a 24-well plate (Corning), and 005 cells ( 1 × 10 ⁴ cells / well) and culture on the plate for 5 days. After 5 days, the culture solution was removed, the cells were washed twice with PBS, 4% (w / v) paraformaldehyde (Wako Pure Chemical Industries, Ltd.) was added, and the cells were fixed at room temperature for 10 minutes. After washing the cells with PBS, PBS containing 0.5% (v / v) Triton X-100 was added and allowed to stand at room temperature for 15 minutes. Then, PBST diluted with 5% (v / v) donkey serum was blocked for 30 minutes at room temperature, and the primary antibody was anti-CD133 antibody (1: 200, rabbit) diluted with PBS containing 1% (v / v) goat serum. Monoclonal antibodies, Proteintech, USA) were used and reacted overnight at 4 ° C. After washing, the cells were immersed in a donkey rabbit antibody (life technology) solution (2 μg / mL) labeled with Alexa-Flor555 for 1 hour at room temperature. After washing, nuclear staining was performed with Hoechst 33258 (Dojindo Laboratories), a preparation was prepared, and then confirmed with a confocal laser microscope (FluoView, Olympus Corporation).

  As a result, it was confirmed that CD133 was expressed and 005 cells were stem cells.

Reference Example 2: Confirmation of differentiation of 005 cells into endothelial cells For 005 cells, expression of PECAM-1 which is an endothelial cell marker was confirmed using an antibody under the following conditions.

005 cells with 2% FBS, 0.4% hydrocortisone, 0.4% hFGF, 0.1% VEGF, 0.1% R3-IGF, 0.1% ascorbic acid, 0.1% hEGF, 0.1% GA-1000, 12 mm cover glass (AGC technograss) coated with poly-L-lysine suspended in EGM®-2 BulletKit® (Lonza, Switzerland) supplemented with 0.1% heparin Strain) was placed in a 24-well plate (Corning), and 005 cells (1 × 10 ⁴ cells / well) were placed therein and cultured on the plate at 1% O ₂ for 7 days. Seven days later, the culture solution was removed, the cells were washed twice with PBS, 4% (w / v) paraformaldehyde (Wako Pure Chemical Industries, Ltd.) was added, and the cells were fixed at room temperature for 10 minutes. After washing the cells with PBS, PBS containing 0.5% (v / v) Triton X-100 was added and allowed to stand at room temperature for 15 minutes. Then, PBST diluted with 5% (v / v) donkey serum was blocked at room temperature for 30 minutes, and anti-PECAM-1 antibody (1: 200, rabbit polyclonal) containing PBST diluted with 5% (v / v) donkey serum. The cover glass was immersed in an antibody (Santa Cruz, USA) solution (5 μg / mL) at 4 ° C. for 16 hours. After washing, the cells were immersed in a donkey rabbit antibody (life technology) solution (5 μg / mL) labeled with Alexa-Fluor 555 at room temperature for 1 hour. After washing, nuclear staining was performed with Hoechst 33258 (Dojindo Laboratories) to prepare a preparation, and then the differentiation of stem cells into endothelial cells was observed with a confocal laser microscope (FluoView, Olympus Corporation).

  The area stained green of 005 cells overlapped with the area stained red with anti-PECAM-1 antibody. From this result, differentiation into endothelial cells was clearly confirmed.

Reference Example 3: Confirmation of tumor stem cell-derived angiogenesis and brain blood vessel-derived angiogenesis in tumors in vivo (a) Preparation of brain tumor model A brain tumor model mouse consists of a cell suspension of 005 cells (1 × 10 ⁴ cells / 3 μL) 1 mm in front of the brain Bregma of nude mice (BALB / c Slc-nu / nu, female, 6-8 weeks old, 15-20 g, Shimizu Experimental Materials Co., Ltd.) It was directly transplanted to a position of 5 mm and a depth of 3 mm.

(B) Preparation of sample and tissue staining After 5 weeks, the mouse brain was removed after perfusion fixation with PBS and 4% (w / v) paraformaldehyde (PFA) (Wako Pure Chemical Industries, Ltd.) Immerse in 4% (w / v) PFA. Thereafter, it was dehydrated by dipping in 30% (w / v) sucrose (Wako Pure Chemical Industries, Ltd.) for 2 days. After dehydration, the brain was frozen and cut to a thickness of 10 μm to prepare a frozen section. The frozen section was washed with PBS, immersed in PBS containing 0.25% (v / v) Triton X-100, and allowed to stand at room temperature for 15 minutes. Then, PBS diluted with 10% (v / v) goat serum was blocked for 60 minutes at room temperature, and anti-PECAM-1 antibody (1: 200, rabbit polyclonal) containing PBS diluted with 1% (v / v) goat serum was used. Antibody, Santa Cruz, USA) solution (5 μg / mL) was soaked at room temperature for 2 hours. After washing, it was immersed in a goat rabbit antibody (life technology) solution (2 μg / mL) labeled with Alexa-Fluor 555 for 1 hour at room temperature. After washing, nuclear staining was performed with Hoechst 33258 (Dojindo Laboratories) to prepare a preparation, and then the in vivo differentiation of stem cells into endothelial cells was observed with a confocal laser microscope (FluoView, Olympus Corporation).

(C) Results A region stained yellow with an anti-PECAM-1 antibody showing tumor blood vessels overlaps with a green stained region showing cells derived from brain tumor stem cells 005 cells. 10-25% was observed.

  This result clearly confirmed tumor stem cell-derived angiogenesis different from normal tissue-derived angiogenesis in tumors.

Reference Example 4: Confirmation that angiogenesis by tumor stem cell-derived endothelial cells does not depend on the VEGF pathway Using 005 cells and HUVEC, the medium was 2% FBS, 0.4% hydrocortisone, 0.4% hFGF, 0 EGM-2 BulletKit with 1% VEGF, 0.1% R3-IGF, 0.1% ascorbic acid, 0.1% hEGF, 0.1% GA-1000, 0.1% heparin (Registered trademark) (Lonza) was cultured according to the protocol and differentiated into vascular endothelial cells.

005 and endothelial cells differentiated from cells (1 × 10 ⁵ cells), 1% sodium dodecyl sulfate was heated to 100 ° C., respectively from vascular endothelial cells differentiated (1 × 10 ⁵ cells) HUVEC (SDS, pure Wako A sample was prepared by dissolving in a solution of Yakuhin Kogyo Co., Ltd. and subjected to polyacrylamide gel electrophoresis.

  Transfer to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Germany) was performed using 48 mM 2-amino-2-hydroxymethylpropane-1,3-diol (Wako Pure Chemical Industries, Ltd.), 39 mM glycine (Japanese 15 V using a semi-dry transfer device (Bio-Rad, USA) in a solution consisting of 1.3 mM SDS, 20 v / v% methanol (Wako Pure Chemical Industries, Ltd.) For 80 minutes. After transfer, the PVDF membrane was placed in 150 mM NaCl containing 5 w / v% skim milk, 0.05 v / v% Tween 20 (MP biomedicals), 20 mM Tris-HCl buffer (pH 7.5, TBST). Shake for 60 minutes at room temperature. The primary antibody was reacted with anti-VEGFR-2 antibody (1: 1000, rabbit monoclonal antibody, Cell Signaling Technology), anti-β-actin antibody (1: 4000, mouse monoclonal antibody, Sigma-Aldrich) at room temperature for 1 hour. It was. After washing 4 times for 3 minutes with TBST, horseradish peroxidase (HRP) -added anti-mouse IgG antibody (1: 4000, Sigma-Aldrich), HRP-added anti-rabbit IgG antibody (1: 4000, Sigma-Aldrich) and 60 at room temperature. Reacted for 1 minute. After washing 4 times for 3 minutes with TBST, it was reacted with HRP chemiluminescence substrate solution ECL Prime (GE Healthcare) for 5 minutes, and the immune complex was detected with a chemiluminescence detector (VersaDoc Model 5000, Bio-Rad). .

  As a result, no band was observed at the position of VEGF-R2 in vascular endothelial cells prepared from 005 cells, and a clear band was observed at the position of VEGF-R2 in vascular endothelial cells prepared from HUVEC.

  As a result of Western blotting, it was proved that VEGF-R2 was not expressed in vascular endothelial cells prepared from 005, and VEGF-R2 was expressed in vascular endothelial cells prepared from HUVEC. Angiogenesis by vascular endothelial cells was shown to be independent of VEGF.

Example 1 Tube Formation Assay 50 μL of growth factor reduced (GFR) Matrigel (Corning) was added to a 96-well plate (Corning) and allowed to gel at 37 ° C. for 60 minutes. 005 cells differentiated into vascular endothelial cells under the same conditions as the differentiation into endothelial cells of Reference Example 2 were seeded at 1.5 × 10 ⁴ cells / 100 μL / well, and the conditions were 37 ° C. and 21% O ₂ . Cultured for 20-24 hours. Observation was performed with an all-in-one fluorescence microscope (KEYENCE). EGM-2 (registered trademark) Bullet Kit (registered trademark) (Lonza) was used as the medium. Control test to confirm tube formation without administration of test drug, and administration of test drug (fluvoxamine, sertraline, paroxetine and imipramine) at each concentration (1 μM, 5 μM and 10 μM) simultaneously with culture A test was conducted to confirm inhibition. FIG. 1 shows fluorescence micrographs observed at predetermined times (0, 6, 20, 24 hours) after cell seeding in the control test. The tube formation inhibition evaluation of each drug was performed by measuring the total length of the tube formed in the visual field for a plurality of visual fields in comparison with the control measurement value in observation at 20 hours after cell seeding. The results are shown in the graph as tube formation inhibition by each test in which the total tube length in the control is 100 (FIGS. 3, 5, 7, and 9). The state of observation of each reagent at 10 μM for 20 hours is shown in FIGS. Fluvoxamine, sertraline and paroxetine were tested simultaneously with the same control, but imipramine was tested separately with the control, so the photo of the control is different from the other examples, but the results were affected. Not give.

As shown in the results, tube formation by tumor stem cell-derived vascular endothelial cells was inhibited in any of the antipsychotic drugs of SSRI (fulboxamine, sertraline, paroxetine) and tricyclic antidepressant (imipramine). The effective concentration (IC ₅₀ ) of each drug is shown in Table 1.

  This result suggests that these antipsychotics inhibit tumor stem cell-derived tumor angiogenesis, as well as all antipsychotic agents (eg, SSRIs, typical antipsychotics, atypical antipsychotics). Drugs, benzodiazepines (BZD), BZD receptor antagonists, tricyclic antidepressants, tetracyclic antidepressants, etc.) are also suggested to inhibit tumor stem cell-derived angiogenesis.

Comparative Example 1: Tube Formation Assay 50 μL of growth factor reduced (GFR) Matrigel (Corning) was added to a 96-well plate (Corning) and gelled at 37 ° C. for 60 minutes. 005 cells or HUVEC differentiated into vascular endothelial cells under the same conditions as the differentiation into endothelial cells of Reference Example 2 were seeded at 1.5 × 10 ⁴ cells / 100 μL / well, and the conditions were 37 ° C. and 21% O ₂ . Incubated for 20-24 hours under. Observation was performed with an all-in-one fluorescence microscope (KEYENCE). EGM-2 (registered trademark) Bullet Kit (registered trademark) was used as the medium. For each cell, a control test for confirming that tubes were formed without administration of the test drug, and inhibition of tube formation by administering the test drug (axitinib) at each concentration (3 nM, 30 nM and 300 nM) simultaneously with the culture. A test was conducted to confirm. The tube formation inhibition evaluation of each drug was performed by measuring the total length of the tube formed in the visual field for a plurality of visual fields in comparison with the control measurement value in observation at 20 hours after cell seeding. The results are shown in the graph as tube formation inhibition by each test where the total tube length in the control is 100 (FIGS. 11 and 13). In addition, the state of observation in 20 hours is shown in FIGS. 10 and 12 for an axitinib concentration of 300 nM, respectively.

  As shown in the results, axitinib showed an inhibitory effect on tube formation by HUVEC, but did not show a significant effect on tube formation by 005 cells, which are tumor stem cells.

Example 2: Concomitant use of fluvoxamine and VEGF pathway inhibitor The antitumor effect of a combination of fluvoxamine and a VEGFR inhibitor, axitinib, which is one of the VEGF pathway inhibitors, was evaluated. As described in (a) of Reference Example 3, a brain tumor model was prepared using nude mice. From 3 weeks after transplantation of 005 cells, axitinib 25 mg / kg (reference administration group n = 3; 0.5 ml as 5% DMSO solution), a combination of axitinib 25 mg / kg and fluvoxamine 50 mg / kg (combination administration group n = 4: 0.5 ml as a 5% DMSO solution (0.25 ml of each solution)) and 5% DMSO as a control (n = 3; 0.5 ml) were intraperitoneally administered once a day. Brain tumor model mice that were administered for 2 weeks were anesthetized with isoflurane, and T2-enhanced images (slice thickness: 0.5 mm, number of slices: 15 sheets, echo time: 22 ms, repetition time: MRI apparatus BioSpec (Bruker, USA): 835 ms) was photographed (FIG. 14A). Thereafter, gadolinium contrast agent (Bayer, Germany) was intraperitoneally administered, and a T1-enhanced image (slice thickness: 0.5 mm, number of slices: 15, echo time: 3.1 ms, repetition time: 235 ms) was taken (FIG. 14). (B)). Both T1 and T2 images were taken in respiratory synchronization. From the MRI image, the tumor volume was calculated by the formula of (major axis x minor axis ² ) / 2. The results are shown in FIG. The values are shown as mean ± standard deviation, and the significance test for each group was performed by Microsoft Office Excel 2013 using t test.

As a result of MRI, a occupying lesion contrasted with gadolinium was observed in the right cerebral hemisphere in both the control and reference administration groups in the T2-Gd image (FIG. 14B). However, no occupying lesion was observed in the group administered with axitinib and fluvoxamine (FIG. 14 (b)). Further, the tumor volume is calculated, the control is 15.2 ± 2.79 mm ^3, the reference administration group 13.3 ± 5.74mm ^3, the combination administration group is 3.23 ± 1.50 mm ^3, and the control A significant difference was observed between the combination administration group (p <0.05). On the other hand, no significant difference was observed between the control and reference administration groups (p = 0.167) (FIG. 15).

  From the above results of MRI images, it can be seen that a significant antitumor effect is exhibited by the combined use of a VEGF pathway inhibitor and an anti-angiogenic agent.

Reference Example 5: Evaluation of target genes by real-time PCR Expression studies of a total of 5 types of genes related to VEGF-independent blood vessel growth were performed. The expression level of mRNA at 6 hours after administration of fluvoxamine (1 μM) was measured by real-time PCR on vascular endothelial cells obtained by differentiating 005 cells under the same conditions as differentiation into endothelial cells of Reference Example 2. Specifically, RNA extraction was performed according to the protocol of RNeasy Mini Kit (Qiagen, Germany), and processing was performed according to the protocol of DNase I (Invitrogen, USA) to remove excess DNA, and SuperScript IV Reverse Transfer transcriptase (Invitrogen, USA). ) And SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, USA). Thereafter, real-time PCR was performed using an apparatus of StepOnePlus Real-Time PCR (Thermo Fisher Scientific, USA) according to the protocol of TaqMan Gene Expression Assays (Applied Biosystems, USA). Real-time PCR was performed in the standard mode and 96-well plate under the conditions shown in Table 2. Gapdh (Mm9999999915_g1) was used as an endogenous control, and target genes were Ang1 (Mm00456503_m1), Fgf-2 (Mm01285715_m1), Dll4 (Mm00444619_m1), Tgfb1 (Mm01178820_m1), and Pdg57 (M011). Further, analysis was performed by the ΔΔCt method based on the Ct value obtained from the result of real-time PCR. The results are shown in FIG.

  Next, vascular endothelial cells derived from the same 005 cells were used, and angiopoietin-1 (Ang1) mRNA involved in vascular endothelial cells and tumor blood vessel growth was real-time before and after administration of fluvoxamine at 6 and 20 hours. Measured by PCR. The results are shown in FIG. From the above results, it was confirmed that the expression level of angiopoietin-1 in the angiopoietin / Tie2 pathway, which is a VEGF-independent pathway, decreased by administration of fluvoxamine in a time-dependent manner.

Reference Example 6: Detection of TIE-2 by Western Blotting Based on the results of Reference Example 5, Western blotting was performed to confirm whether TIE2, which is an ANG receptor, was originally expressed in tumor blood vessels derived from brain tumor stem cells. It was. In addition, the expression of VEGFR2, which is a receptor for VEGF, which is a blood vessel growth factor, was also confirmed. For 005 cells (stem cells), brain tumor vascular endothelial cells (TDEC) derived from brain tumor stem cells obtained by differentiating 005 cells under the same conditions as the differentiation into endothelial cells of Reference Example 2, 4 days after changing to the endothelial cell differentiation medium Cells at day 7 and day 7 were dissolved in 1% sodium dodecyl sulfate (SDS, Wako Pure Chemical Industries, Ltd.) solution heated to 100 ° C. each as HUVEC as a positive control. Electrophoresis was performed.

  For transfer to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Germany), 48 mM 2-amino-2-hydroxymethylpropane-1,3-diol (Wako Pure Chemical Industries, Ltd.), 39 mM glycine ( In a solution consisting of Wako Pure Chemical Industries, Ltd., 1.3 mM SDS, 20% (v / v) methanol (Wako Pure Chemical Industries, Ltd.), a semi-dry transfer device (Bio-Rad, USA) was used. For 15 minutes at 15V. After the transfer, the PVDF membrane was subjected to 150 mM Nacl containing 5% (w / v) skim milk, 0.05% (v / v) Tween 20 (MP Biomedicals), 20 mM Tris-HCl buffer (pH 7.5, TBST). Shake for 1 hour at room temperature. As primary antibodies, anti-VEGFR-2 antibody (1: 1000, rabbit monoclonal antibody, Cell Signaling Technology, USA), anti-TIE-2 antibody (1: 1000, rabbit monoclonal antibody, Proteintech), anti-β-actin antibody (1 : 4000, mouse monoclonal antibody, Sigma-Aldrich) at room temperature for 1 hour. After washing 5 times with TBST for 5 minutes, horseradish peroxidase (HRP) -added anti-mouse IgG antibody (1: 4000, Sigma-Aldrich), HRP-added anti-rabbit IgG antibody (1: 4000, Sigma-Aldrich) and 1 at room temperature. Reacted for hours. After washing 5 times with TBST for 5 minutes, it was reacted with HRP chemiluminescence substrate solution ECL Prime (GE Healthcare) for 5 minutes, and the immune complex was detected with a chemiluminescence detector (VersaDoc Model 5000, Bio-Rad).

  We first examined VEGFR2 which plays a central role in angiogenesis in brain tumor stem cell-derived brain tumor vascular endothelial cells (TDEC) and cerebral blood vessel-derived vascular endothelial cells (HUVEC). Similar to Reference Example 4, almost no expression of VEGFR2 could be confirmed in TDEC, while strong expression of VEGFR2 was observed in HUVEC. Next, the expression of TIE2, which is a VEGF-independent pathway, was examined. It was revealed that TIE2 was expressed in TDEC differentiated from 005. Moreover, the expression of TIE2 was also confirmed in HUVEC (FIG. 17).

Claims (7)

Tumor stem cell-derived angiogenesis inhibitors including antipsychotics. The tumor stem cell-derived angiogenesis inhibitor according to claim 1, wherein the antipsychotic is at least one selected from a selective serotonin reuptake inhibitor (SSRI) or a tricyclic antidepressant. The tumor stem cell-derived angiogenesis inhibitor according to claim 1 or 2, wherein the antipsychotic drug is SSRI. The tumor stem cell-derived angiogenesis inhibitor according to claim 2, wherein the antipsychotic is at least one selected from the group consisting of fluvoxamine, sertraline, paroxetine and imipramine. The tumor stem cell-derived angiogenesis inhibitor according to any one of claims 1 to 4, which is used for treatment of VEGF pathway inhibitor resistant cancer. The cancer therapeutic agent which combined the tumor stem cell origin angiogenesis inhibitor and VEGF pathway inhibitor of any one of Claims 1-5. The cancer therapeutic agent according to claim 6, which is used for treatment of VEGF pathway inhibitor resistant cancer.