WO2024202110A1 - Anticancer agent - Google Patents
Anticancer agent Download PDFInfo
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- WO2024202110A1 WO2024202110A1 PCT/JP2023/032557 JP2023032557W WO2024202110A1 WO 2024202110 A1 WO2024202110 A1 WO 2024202110A1 JP 2023032557 W JP2023032557 W JP 2023032557W WO 2024202110 A1 WO2024202110 A1 WO 2024202110A1
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- erythritol
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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
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
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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
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
- A61K31/047—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates having two or more hydroxy groups, e.g. sorbitol
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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
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/194—Carboxylic acids, e.g. valproic acid having two or more carboxyl groups, e.g. succinic, maleic or phthalic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/243—Platinum; Compounds thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- the present invention relates to a pharmaceutical composition that inhibits cancer growth and metastasis.
- Cancer is a general term for cells that grow without autonomous control in the body. It is a disease caused by genetic mutations in normal cells, and is broadly divided into solid cancers, including carcinomas that develop in epithelial cells and sarcomas that develop in non-epithelial cells, and blood cancers that originate in blood cells. In Japan, the cumulative risk of developing cancer in a lifetime is 65.5% for men and 51.2% for women, and it is said that one in two people will develop some form of cancer in their lifetime (National Cancer Center Cancer Information Service, 2019 data).
- Cancer treatment methods mainly include surgery (surgical treatment), drug therapy (chemotherapy), and radiation therapy, as well as multidisciplinary treatments that combine these therapies.
- Drug therapy is a treatment that uses anticancer drugs to prevent cell proliferation, and is used to suppress cancer proliferation and prevent metastasis and recurrence. While surgery and radiation therapy are localized treatments for cancer, anticancer drugs have a wider range of therapeutic effects, making them an effective treatment when there is a possibility of metastasis or when wide-area treatment is required, such as in the case of blood cancer.
- Anticancer drugs include cytotoxic anticancer drugs such as DNA synthesis inhibitors, microtubule inhibitors, and antibiotics, molecular targeted drugs that target specific molecules such as kinases involved in the proliferation of cancer cells, and endocrine therapy drugs that use hormones to inhibit the secretion and action of hormones.
- cytotoxic anticancer drugs such as DNA synthesis inhibitors, microtubule inhibitors, and antibiotics
- molecular targeted drugs that target specific molecules such as kinases involved in the proliferation of cancer cells
- endocrine therapy drugs that use hormones to inhibit the secretion and action of hormones.
- all drugs act on normal cells as well as cancer cells, causing a number of side effects. The effects on normal cells vary depending on the drug used, but examples include hair loss, pancytopenia, nausea, and vomiting.
- treatments have been developed to reduce side effects of pancytopenia, such as the administration of a drug (G-CSF: granulocyte colony-stimulating factor) that stimulates bone marrow stem cells
- Patent Documents 1 and 2 disclose methods and drugs to reduce side effects.
- Patent Document 1 discloses a side effect reduction agent that reduces the side effects of anticancer drugs in specific amino acids and amino acid derivatives and improves the completion rate of chemotherapy treatment.
- Patent Document 2 discloses an administration plan support device that acquires the symptoms and onset time of side effects of individual patients as biometric information, predicts when side effects will occur the next time a therapeutic drug is administered, and uses the information to help treat side effects.
- Patent Document 1 increases the rate of treatment completion, it only suppresses the onset of certain side effects such as stomatitis, loss of appetite, and nausea, and its effectiveness is limited.
- the invention described in Patent Document 2 is a support device that predicts side effects during the next treatment based on side effects during the first treatment and proposes a dosing plan with fewer side effects, so it cannot be used for the first treatment and does not lead to a fundamental reduction in side effects. Therefore, there is a demand for the development of a pharmaceutical composition that has fewer side effects and has anti-cancer effects.
- the present invention aims to provide a pharmaceutical composition that can effectively inhibit the proliferation and metastasis of cancer cells without side effects.
- Erythritol is a type of sugar alcohol that is also naturally contained in vegetables and fruits. Industrially, it is a zero-calorie sweetener produced by fermenting glucose with yeast, and is a highly safe ingredient that is widely used in foods that promote reduced calories. Erythritol is also an ingredient used in cosmetics, health foods, and general foods that are commonly distributed, and is a compound whose safety has already been proven. The inventors discovered that erythritol has anti-cancer effects and completed the present invention. Furthermore, when compounds with similar structures were analyzed, it was found that xylitol and mesotartaric acid also have anti-cancer effects.
- Non-Patent Document 1 discloses that, using the brain tumor cell line U87, cell proliferation was inhibited in the presence of high concentrations of erythritol.
- Non-Patent Document 1 presents the results of an experiment using a cell line, and does not disclose the route of administration. Since it is well known that the effects of anticancer drugs differ depending on the route of administration, the route of administration was investigated. As a result, it was revealed that erythritol is effective when administered parenterally.
- the present invention relates to the following pharmaceutical compositions: (1) An anticancer agent having erythritol, xylitol, or mesotartaric acid as an active ingredient.
- the cell proliferation effect was observed in the sugar alcohols erythritol and xylitol, and the organic acid mesotartaric acid.
- Erythritol and xylitol are sugar alcohols that have traditionally been used in food and cosmetics. As the safety of these compounds is guaranteed, they are expected to be used as anticancer drugs with few side effects.
- the anticancer agent according to (2) characterized in that it contains erythritol as an active ingredient.
- erythritol in particular has been confirmed to be effective not only in vitro but also in vivo, and is therefore believed to function as a useful anticancer agent.
- the anticancer agent according to (3) which is used in combination with another anticancer agent.
- Conventionally used anticancer drugs have side effects that have been problematic, but erythritol, when used in combination with other anticancer drugs, has the effect of reducing the IC50 of the other anticancer drugs. In other words, it is possible to treat cancer by reducing the amount of anticancer drugs that have strong side effects.
- a method for treating a cancer patient comprising parenterally administering an anticancer agent containing erythritol as an active ingredient.
- FIG. 1 shows the cell proliferation inhibitory effect of adding erythritol.
- FIG. 1 shows the results of analyzing the ATP production ability of cells by addition of erythritol.
- FIG. 1 shows the results of RNAseq analysis of changes in gene expression due to the addition of erythritol.
- FIG. 1 shows the results of analyzing changes in gene expression by pathway analysis.
- FIG. 1 shows the results of intravenously administering erythritol to cancer model mice and analyzing the effect of inhibiting cancer cell proliferation.
- FIG. 1 shows the results of orally administering erythritol to cancer model mice and analyzing the effect on cancer cell proliferation.
- FIG. 1 shows the results of an analysis of the migration ability of cancer cells with the addition of erythritol.
- FIG. 1 shows the results of an analysis of the cell proliferation inhibitory effects of various sugar alcohols.
- FIG. 1 shows the structures of the analyzed compounds.
- FIG. 1 shows the results of analyzing the cell proliferation inhibitory effects of L-threitol and mesotartaric acid.
- the anticancer agent of the present invention can be used regardless of the type of cancer.
- the cancer may be, but is not limited to, sarcoma, leukemia, biliary tract cancer, breast cancer, uterine cancer, colorectal cancer, laryngeal cancer, esophageal cancer, stomach cancer, colon cancer, tonsil cancer, tongue cancer, neck cancer, lymphoma, lung cancer, thyroid cancer, ovarian cancer, kidney cancer, pancreatic cancer, brain tumor, myeloma, glioma, melanoma, liver cancer, prostate cancer, and bladder cancer.
- the agent inhibits the migration ability of cancer cells, and is therefore believed to act not only to inhibit cancer proliferation, but also to inhibit metastasis.
- the pharmaceutical of the present invention is ineffective when administered orally, but is effective when administered parenterally. Therefore, it can be administered parenterally, for example, intravenously, intramuscularly, subcutaneously, intraspinally or intradermally, as an injection or drip infusion, with intravenous administration being preferred. There is also no limit to the number of times it is administered, but it can be administered once or several times a day, or at intervals.
- the anticancer agent of the present invention is used as an injection as described above, when it is provided in a liquid form, it is provided in a form dissolved in water, physiological saline, etc., or as a lyophilized injection obtained by lyophilization together with an excipient, or as a powder preparation obtained by crystallization, etc.
- it may contain a preservative, a solubilizer, a stabilizer, a wetting agent, an emulsifier, a salt that changes the osmotic pressure, a buffering agent, or an antioxidant.
- the anticancer agent of the present invention reduces the IC 50 of other anticancer agents when used in combination with other anticancer agents, particularly cytotoxic anticancer agents. Therefore, it can be provided in an embodiment that contains other anticancer agents.
- the effective amount or dosage of the compound of the present invention may be appropriately selected depending on the administration form, age, body weight, and symptoms, and is not particularly limited, but can be administered at a concentration ranging from 8.2 mM to 245.7 mM, as shown in the following examples.
- Example 1 [Erythritol inhibits the proliferation of cancer cells]
- Mouse lung cancer cells, Lewis lung carcinoma (LLC) cells (RIKEN BRC CELL BANK) were seeded at 5 x 103 cells/well in a 96-well plate (BM Equipment Co., Ltd.), and erythritol was added to 90 ⁇ L of DMEM medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), and the cells were cultured for 24 and 48 hours.
- LLC Lewis lung carcinoma
- FBS fetal bovine serum
- Erythritol (Itochu Foods Co., Ltd.) added to the medium was dissolved in phosphate buffered saline (PBS) at 0.082, 0.819, and 2.457 M, respectively, and sterilized using a 0.2 mm (ADVANTEC) filter, and then added to the medium. That is, the final concentrations were 8.2 mM, 81.9 mM, and 245.7 mM.
- PBS phosphate buffered saline
- Cell proliferation was evaluated using cell counting kit-8 (Dojindo Molecular Technologies). After 24 and 48 hours of culture, 10 ⁇ L of tetrazolium salt WST-8 was added, the cells were left to stand at 37°C for 1 hour, and the absorbance at 450 nm was measured. The calculated values were calculated by taking the average of each absorbance and then calculating the relative value with the control value set at 1.0 ( Figure 1).
- Erythritol (shown as ERT in Figure 1) was added to the medium at final concentrations ranging from 8.2 mM to 245.7 mM, and cell proliferation was measured after 24 and 48 hours. As a result, after 24 hours, the addition of erythritol at a concentration of 81.9 mM or higher significantly (p ⁇ 0.05) inhibited cell proliferation, and after 48 hours, even at 8.2 mM (p ⁇ 0.01). It was also shown that the inhibition of cell proliferation by erythritol is concentration-dependent. In the figures below, * indicates p ⁇ 0.05, ** indicates p ⁇ 0.01, and *** indicates p ⁇ 0.001.
- Example 2 [Effect of erythritol on gene expression related to cancer cell proliferation] Analysis of changes in gene expression after the addition of erythritol was performed. 245.7 mM erythritol and an equal amount of PBS as a control were added to the LLC cell medium, and 48 hours later, the cells were extracted using miRNeasy Kits (QIAGEN), and RNA-seq was performed at Bioengineering Lab Co., Ltd. The obtained data was analyzed using CLC Genomics Workbench (CLC bio) and Gene Set Enrichment Analysis (GSEA) (UC San Diego and Broad Institute).
- CLC Genomics Workbench CLC bio
- GSEA Gene Set Enrichment Analysis
- erythritol was dissolved in saline at 2.5M, and 0.2 mL was administered from the tail vein using a needle-equipped syringe (My Shot, 30G x 13mm, NIPRO) (single dose, 60 mg, human equivalent dose 162.6 mg/kg). Erythritol was injected into the tail vein once per day. During the experiment, the extent of cancer colonization was measured using a gauge, and the animals were dissected after 2 weeks. The size of the cancer (mm 3 ) was calculated by 0.4 (coefficient) x (minimum diameter mm x maximum diameter mm), and the average value was calculated (Figure 5). Since there are no side effects, no upper limit for the dosage is specified.
- Erythritol administration tended to suppress the spread of cancer, and compared to the control (PBS), cancer spread tended to be delayed in mice administered erythritol.
- the average body weight was 18.9g in the control group and 19.1g in the erythritol-administered group, and before dissection it was 19.5g in the control group and 20.3g in the erythritol-administered group.
- Kestose is a carbohydrate with a completely different structure, and serves as a control.
- 1 x 106 LLC cells were diluted with 0.2 mL of physiological saline and injected subcutaneously into the right groin of the mice using a needle-equipped syringe. After that, the mice were continuously fed each diet, and the tumor colonization was measured daily using a gauge, and the mice were dissected after 2 weeks. The size of the tumor was measured, and the average tumor volume was calculated (Figure 6). No significant difference in tumor volume was observed during the experimental period in any of the control group, erythritol-administered group, and kestose-administered group. It was revealed that erythritol has no effect on tumor growth when administered orally, and is only effective when administered parenterally.
- Example 4 [Effect of erythritol on cancer cell migration] From the results of Example 2, the addition of erythritol changed gene expression related to cancer invasion and metastasis, so the effect on cell migration was analyzed. 400 ⁇ L of DMEM medium containing 0.1% FBS was placed in an insert (Culture insert for 24 wells, 0.4 ⁇ m PET transparent membrane, Falcon), and LLC cells were seeded on the insert at a cell count of 1 ⁇ 10 5 cells. 1000 ⁇ L of serum-free DMEM medium to which erythritol was added to make 245.7 mM was placed in a 24-well cell culture insert companion plate (Corning), and cultured for 24 hours.
- the insert was removed, the medium was aspirated, and the inside of the insert was wiped with a cotton swab.
- the insert was transferred to a well containing 1 mL of 70% ethanol, and 0.5 mL of 70% ethanol was added to the insert. After leaving it for 30 minutes, the 70% ethanol in the insert was removed by aspiration, and the insert was transferred to a new 24-well containing 1 mL of PBS. 0.5 mL of PBS was added to the insert to wash it. After washing twice, the PBS in the insert was removed by aspiration.
- Giemsa (Sigma-Aldrich) was diluted 10-fold with pure water to prepare the Giemsa staining solution.
- 24 wells containing 1 mL of Giemsa staining solution were prepared, inserts were placed in them, and 0.5 mL of Giemsa staining solution was added to the inserts. After leaving it for 30 minutes, the Giemsa staining solution in the inserts was removed by suction. 24 new wells containing 1 mL of pure water were prepared, and the inserts were placed in them. 0.5 mL of pure water was added to the inserts to wash them. After washing twice, images were taken under a microscope (all-in-one fluorescent microscope, Keyence). The number of cells was counted from the images and compared to the control ( Figure 7).
- erythritol In the group to which erythritol was added, the migration ability of cells was significantly suppressed (p ⁇ 0.01). This result suggests that erythritol may inhibit the ability of cells to migrate, that is, inhibit the invasion and metastasis of cancer cells.
- PBS + 0.1% triton was removed by aspiration, and the cover glass was washed with 2 mL of PBS. PBS was removed by aspiration, and washing was performed again. Actin staining was performed with Alexa Fluor 488-labeled Phalloidin (1:100, Thermo Fisher), and nuclear staining was performed with DAPI (1:35000, Abcam) diluted with 1% bovine serum albumin (BSA) and stained for 1 hour. The cover glass was washed three times with 1 mL PBS containing 0.1% Tween, and then washed three times with 1 mL PBS. The glass was mounted with 10 ⁇ L fluromount (Diagnostic Biosystems), left for 1 hour, and then photographed under a microscope (Keyence) (FIG. 8).
- Example 6 [Effects of various sugar alcohols on cancer cells and their combination with anticancer drugs] Since erythritol was found to have a tumor cell proliferation inhibitory effect, the possibility that other sugar alcohols may have a similar effect was considered, and in addition to erythritol, xylitol and sorbitol were also analyzed for their tumor proliferation inhibitory effects. LLC cells were seeded at 5 x 103 cells in a 96-well microtiter plate (BM Equipment Co., Ltd.) using DMEM medium containing 10% FBS, and pre-cultured for 24 hours.
- BM Equipment Co., Ltd. BM Equipment Co., Ltd.
- erythritol (ERT), xylitol (XRT), and sorbitol (SRT) were dissolved in PBS to final concentrations of 8.2 mM, 81.9 mM, and 245.7 mM, respectively, and sterilized using a 0.2 mm (ADVANTEC) filter, and then added to the medium.
- the liquid volume was 9 ⁇ l/well.
- cisplatin was dissolved in dimethyl sulfoxide (Wako) as an anticancer drug and added at concentrations of 0 to 40 ⁇ M. The amount of liquid added was 1 ⁇ l/well.
- Cell counting kit-8 was used for the evaluation. 10 ⁇ L of water-soluble tetrazolium salt WST-8 was added, and the cells were left to stand at 37°C for 1 hour, and absorbance was measured at a wavelength of 450 nm. The calculated values were calculated by taking the average of each absorbance, and then calculating the relative value with the control value set at 1.0.
- the concentration of cisplatin at which the number of cells was halved at each time point was evaluated (Figure 9).
- sorbitol was no different from the control at 8.2 mM and 82 mM, and only the effect of cisplatin was observed.
- erythritol and xylitol were used in combination with cisplatin, they were shown to additively inhibit cell proliferation at concentrations of 82 mM and 245.7 mM.
- LLC cells were seeded at 5 x 103 cells/well in a 96-well plate, and L-threitol (Sigma-Aldrich) or mesotartaric acid (Fujifilm Wako) dissolved in PBS at 8.2, 81.9, or 245.7 mM was added to the medium at 1/10 volume, followed by incubation for 24 and 48 hours. As a control, an equal volume of the solvent PBS was added. Cell proliferation was evaluated using cell counting kit-8. The results are shown in FIG. 11.
- erythritol, xylitol, and mesotartaric acid have cell proliferation inhibitory effects, and among these, erythritol has a concentration-dependent cell proliferation inhibitory effect. Further analysis showed that erythritol not only inhibits cell proliferation, but also inhibits migration, and is therefore considered to have an inhibitory effect on cancer metastasis. Erythritol has traditionally been used in foods and cosmetics, and anticancer drugs that contain it as an active ingredient are thought to be highly safe and have no side effects.
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Abstract
Description
本発明はがんの増殖や転移を抑制する医薬組成物に関する。 The present invention relates to a pharmaceutical composition that inhibits cancer growth and metastasis.
がんとは、生体内の自律的制御をうけず増殖を行う細胞の総称である。正常な細胞に遺伝子の変異が生じることによる疾患であり、上皮細胞から発生する癌腫や非上皮性細胞から発生する肉腫を含む固形がんと、血球に由来する血液がんに大別される。日本では一生のうちにがんに罹患する累積罹患リスクは、男性では６５．５％、女性では５１．２％であり、２人に１人が一生のうちに何らかのがんにかかると言われている（国立がん研究センターがん情報サービス、２０１９年データ）。 Cancer is a general term for cells that grow without autonomous control in the body. It is a disease caused by genetic mutations in normal cells, and is broadly divided into solid cancers, including carcinomas that develop in epithelial cells and sarcomas that develop in non-epithelial cells, and blood cancers that originate in blood cells. In Japan, the cumulative risk of developing cancer in a lifetime is 65.5% for men and 51.2% for women, and it is said that one in two people will develop some form of cancer in their lifetime (National Cancer Center Cancer Information Service, 2019 data).
がんは、発見が遅れ進行が進んだ場合には、死亡につながるため、治療方法の確立が重要な疾病であるといえる。がんの治療法には主として、手術（外科治療）、薬物療法（化学療法）、放射線治療があり、さらに、これら治療法を組み合わせて治療する集学的治療がある。薬物療法は、細胞の増殖を防ぐ抗がん剤を用いた治療法で、がんの増殖を抑制したり、転移や再発を防ぐために使用する薬剤である。手術や放射線治療が、がんに対する局所的な治療であるのに対し、抗がん剤は、より広い範囲に治療の効果が及ぶことから、転移の可能性があるときや、血液がんのように広い範囲での治療が必要である場合に有効な治療法である。 Since cancer can lead to death if it is discovered late and allowed to progress, it is a disease for which it is important to establish treatment methods. Cancer treatment methods mainly include surgery (surgical treatment), drug therapy (chemotherapy), and radiation therapy, as well as multidisciplinary treatments that combine these therapies. Drug therapy is a treatment that uses anticancer drugs to prevent cell proliferation, and is used to suppress cancer proliferation and prevent metastasis and recurrence. While surgery and radiation therapy are localized treatments for cancer, anticancer drugs have a wider range of therapeutic effects, making them an effective treatment when there is a possibility of metastasis or when wide-area treatment is required, such as in the case of blood cancer.
抗がん剤には、ＤＮＡ合成阻害剤、微小管阻害薬、抗生物質等の細胞傷害性抗がん剤、がん細胞の増殖に関与するキナーゼ等、特定の分子を標的とする分子標的薬、ホルモンを利用してホルモンの分泌や作用を阻害する内分泌療法薬などがある。しかし、いずれの薬剤も、がん細胞と同様に正常細胞にも作用するため副作用が少なからず生じる。正常細胞の受ける影響は、使用する薬剤により様々であるが、例えば、脱毛、汎血球減少、吐き気・嘔吐などがある。汎血球減少に対しては、骨髄幹細胞に血球の増産を働きかける薬剤（Ｇ－ＣＳＦ：顆粒球コロニー刺激因子）の投与など、副作用を軽減する治療法が開発されてきているものの、すべての副作用に対応できるわけではない。そこで、副作用を軽減する方法、治療薬が提案されている（特許文献１、２）。特許文献１には、特定のアミノ酸及びアミノ酸誘導体に抗がん剤の副作用を軽減し、化学療法の治療完遂率を改善する副作用軽減剤が開示されている。特許文献２には、個々の患者の副作用の症状や発生時期を生体情報として取得し、次回の治療薬投与時の副作用の発生時期等を予測し、副作用の治療に役立てようとする投与計画支援装置が開示されている。
Anticancer drugs include cytotoxic anticancer drugs such as DNA synthesis inhibitors, microtubule inhibitors, and antibiotics, molecular targeted drugs that target specific molecules such as kinases involved in the proliferation of cancer cells, and endocrine therapy drugs that use hormones to inhibit the secretion and action of hormones. However, all drugs act on normal cells as well as cancer cells, causing a number of side effects. The effects on normal cells vary depending on the drug used, but examples include hair loss, pancytopenia, nausea, and vomiting. Although treatments have been developed to reduce side effects of pancytopenia, such as the administration of a drug (G-CSF: granulocyte colony-stimulating factor) that stimulates bone marrow stem cells to increase blood cell production, this does not cover all side effects. Therefore, methods and drugs to reduce side effects have been proposed (
しかしながら、特許文献１に記載の発明は、治療完遂率を上げるものの、口内炎、食欲不振、悪心等、特定の副作用の発症を抑えるものであり、その効果は限定的である。特許文献２に記載の発明は、初回治療時の副作用から、次回治療時における副作用を予測し、副作用の少ない投与計画を提案する支援装置であるため、初回治療には対応できず、根本的な副作用の軽減につながるものではない。そのため、副作用自体が少なく、抗がん効果を有する医薬組成物の開発が求められている。
However, although the invention described in
本発明は、副作用を伴わず、効果的にがん細胞の増殖と転移を抑制することができる医薬組成物を提供することを課題とする。エリスリトールは、天然の野菜や果物にも含有されている糖アルコールの一種であり、工業的にはブドウ糖から酵母を用いて発酵させることにより製造されるゼロカロリーの甘味料であり、広くカロリー低減を訴求した食品に配合される安全性の高い成分である。エリスリトールは、一般流通している化粧品や健康食品や一般食品においても配合されている成分であり、安全性が高いことはすでに証明されている化合物である。本発明者らは、エリスリトールに抗がん作用があることを見出し、本発明を完成させた。さらに、構造の類似した化合物を解析したところ、キシリトール、メソ酒石酸にも抗がん作用があることを見出した。 The present invention aims to provide a pharmaceutical composition that can effectively inhibit the proliferation and metastasis of cancer cells without side effects. Erythritol is a type of sugar alcohol that is also naturally contained in vegetables and fruits. Industrially, it is a zero-calorie sweetener produced by fermenting glucose with yeast, and is a highly safe ingredient that is widely used in foods that promote reduced calories. Erythritol is also an ingredient used in cosmetics, health foods, and general foods that are commonly distributed, and is a compound whose safety has already been proven. The inventors discovered that erythritol has anti-cancer effects and completed the present invention. Furthermore, when compounds with similar structures were analyzed, it was found that xylitol and mesotartaric acid also have anti-cancer effects.
エリスリトールの腫瘍抑制効果に関しては、すでに報告がある。非特許文献１には、脳腫瘍細胞株Ｕ８７を用いて、高濃度のエリスリトール存在下で細胞増殖阻害が認められたことが開示されている。しかし、非特許文献１は、細胞株を用いた実験結果であり、その投与経路については記載がない。投与経路によって抗がん剤の効果が異なることは良く知られていることであるから、投与経路について検討を行った。その結果、エリスリトールは、非経口投与を行った場合に効果を奏することを明らかにした。
The tumor-suppressing effect of erythritol has already been reported. Non-Patent
本発明は、以下の医薬組成物に関する。
（１）エリスリトール、キシリトール、又はメソ酒石酸を有効成分とする抗がん剤。
糖アルコールであるエリスリトール、キシリトール、また有機酸であるメソ酒石酸に、細胞増殖効果が認められた。エリスリトール、キシリトールは、従来から食品や化粧品に用いられている糖アルコールである。これら化合物は、安全性が担保されていることから、副作用の少ない抗がん剤として使用が期待できる。
The present invention relates to the following pharmaceutical compositions:
(1) An anticancer agent having erythritol, xylitol, or mesotartaric acid as an active ingredient.
The cell proliferation effect was observed in the sugar alcohols erythritol and xylitol, and the organic acid mesotartaric acid. Erythritol and xylitol are sugar alcohols that have traditionally been used in food and cosmetics. As the safety of these compounds is guaranteed, they are expected to be used as anticancer drugs with few side effects.
（２）がんの増殖を抑制するものであることを特徴とする（１）記載の抗がん剤。
以下の実施例で示すが、ｉｎ ｖｉｔｒｏの実験からこれら化合物はがん細胞の増殖抑制効果あることが示された。
(2) The anticancer agent according to (1), which is characterized by suppressing cancer proliferation.
As shown in the following examples, in vitro experiments have demonstrated that these compounds have the effect of inhibiting the proliferation of cancer cells.
（３）エリスリトールを有効成分とするものであることを特徴とする（２）記載の抗がん剤。
これら化合物のうち、特に、エリスリトールはｉｎ ｖｉｔｒｏだけではなく、ｉｎ ｖｉｖｏでの効果も確認されていることから、有用な抗がん剤として機能するものと考えられる。
(3) The anticancer agent according to (2), characterized in that it contains erythritol as an active ingredient.
Among these compounds, erythritol in particular has been confirmed to be effective not only in vitro but also in vivo, and is therefore believed to function as a useful anticancer agent.
（４）がんの転移を抑制するものである（３）記載の抗がん剤。
また、エリスリトールは細胞の遊走能も抑制することから、がんの転移を抑制するものと推認される。
(4) The anticancer agent according to (3), which suppresses cancer metastasis.
Erythritol also inhibits cell migration, and is therefore presumed to inhibit cancer metastasis.
（５）非経口投与であることを特徴とする（３）記載の抗がん剤。
非経口投与、特に血中投与で有効であることが確認された。
(5) The anticancer agent according to (3), which is administered parenterally.
It has been confirmed that it is effective when administered parenterally, particularly when administered into the blood.
（６）他の抗がん剤と併用することを特徴とする（３）記載の抗がん剤。
従来から用いられている抗がん剤はその副作用が問題となっているが、エリスリトールは、抗がん剤と併用することによって、他の抗がん剤のＩＣ５０を減少させる効果があった。すなわち、副作用の高い抗がん剤を減らして治療することが可能となる。
(6) The anticancer agent according to (3), which is used in combination with another anticancer agent.
Conventionally used anticancer drugs have side effects that have been problematic, but erythritol, when used in combination with other anticancer drugs, has the effect of reducing the IC50 of the other anticancer drugs. In other words, it is possible to treat cancer by reducing the amount of anticancer drugs that have strong side effects.
（７）他の抗がん剤が細胞傷害性抗がん剤であることを特徴とする（６）記載の抗がん剤。
特に、副作用が強くでる細胞傷害性抗がん剤で相加効果が認められたことは、既存の医薬と併用するうえで非常に意味がある。
(7) The anticancer agent according to (6), wherein the other anticancer agent is a cytotoxic anticancer agent.
In particular, the fact that an additive effect was observed with cytotoxic anticancer drugs, which have strong side effects, is extremely significant when used in combination with existing medications.
（８）がん患者の治療方法であって、エリスリトールを有効成分とする抗がん剤を非経口投与することを特徴とする治療方法。
（９）前記非経口投与が静脈投与であることを特徴とする（８）記載の治療方法。
（１０）他の抗がん剤を併用投与することを特徴とする（８）又は（９）記載の治療方法。
（１１）前記他の抗がん剤が、細胞障害性抗がん剤であることを特徴とする（１０）記載の治療方法。
(8) A method for treating a cancer patient, comprising parenterally administering an anticancer agent containing erythritol as an active ingredient.
(9) The method of treatment according to (8), wherein the parenteral administration is intravenous administration.
(10) The method of treatment according to (8) or (9), characterized in that another anticancer drug is administered in combination.
(11) The method of treatment according to (10), wherein the other anticancer drug is a cytotoxic anticancer drug.
本発明の抗がん剤は、がんの種類によらず使用することができる。例えば、がんは以下に限定されるものではないが、肉腫、白血病、胆道がん、乳がん、子宮がん、結腸直腸がん、喉頭がん、食道がん、胃がん、大腸がん、扁桃がん、舌がん、首のがん、リンパ腫、肺がん、甲状腺がん、卵巣がん、腎がん、すい臓がん、脳腫瘍、骨髄腫、神経膠腫、メラノーマ、肝がん、前立腺がん及び膀胱がんが例示される。また、以下で説明するが、がん細胞の遊走能を抑制することから、がんの増殖抑制だけではなく、転移抑制にも作用するものと考える。 The anticancer agent of the present invention can be used regardless of the type of cancer. For example, the cancer may be, but is not limited to, sarcoma, leukemia, biliary tract cancer, breast cancer, uterine cancer, colorectal cancer, laryngeal cancer, esophageal cancer, stomach cancer, colon cancer, tonsil cancer, tongue cancer, neck cancer, lymphoma, lung cancer, thyroid cancer, ovarian cancer, kidney cancer, pancreatic cancer, brain tumor, myeloma, glioma, melanoma, liver cancer, prostate cancer, and bladder cancer. As will be explained below, the agent inhibits the migration ability of cancer cells, and is therefore believed to act not only to inhibit cancer proliferation, but also to inhibit metastasis.
本発明の医薬は、経口投与では効果がなく、非経口的に投与することによって効果を生じる。したがって、注射剤、あるいは点滴剤として非経口的、例えば静脈内、筋肉内、皮下、脊髄内又は皮内的に投与することができ、静脈内投与であることが好ましい。また、投与回数も限定されるものではないが、１日に１回または数回、あるいは投与間隔をあけて投与できる。 The pharmaceutical of the present invention is ineffective when administered orally, but is effective when administered parenterally. Therefore, it can be administered parenterally, for example, intravenously, intramuscularly, subcutaneously, intraspinally or intradermally, as an injection or drip infusion, with intravenous administration being preferred. There is also no limit to the number of times it is administered, but it can be administered once or several times a day, or at intervals.
本発明の抗がん剤は、上述のように注射剤として用いることから、液剤で提供される場合には、水、生理食塩水等に溶解した形態で、あるいは、賦形剤とともに凍結乾燥した凍結乾燥注射剤、あるいは晶析等により得た粉末製剤として提供される。また、防腐剤、可溶化剤、安定剤、湿潤剤、乳化剤、浸透圧を変える塩、緩衝剤又は酸化防止剤などを含んでよい。また、本発明の抗がん剤は、他の抗がん剤、特に細胞傷害性の抗がん剤と併用すると、他の抗がん剤のＩＣ５０を低下させることが示された。したがって、他の抗がん剤を含む態様で提供することができる。 Since the anticancer agent of the present invention is used as an injection as described above, when it is provided in a liquid form, it is provided in a form dissolved in water, physiological saline, etc., or as a lyophilized injection obtained by lyophilization together with an excipient, or as a powder preparation obtained by crystallization, etc. In addition, it may contain a preservative, a solubilizer, a stabilizer, a wetting agent, an emulsifier, a salt that changes the osmotic pressure, a buffering agent, or an antioxidant. In addition, it has been shown that the anticancer agent of the present invention reduces the IC 50 of other anticancer agents when used in combination with other anticancer agents, particularly cytotoxic anticancer agents. Therefore, it can be provided in an embodiment that contains other anticancer agents.
本発明の化合物の有効量または投与量は、投与形態、年齢、体重、症状に応じて適宜選択すればよく、特に制限されないが、以下の実施例で示しているように、８．２ｍＭから２４５．７ｍＭまでの濃度で投与することができる。 The effective amount or dosage of the compound of the present invention may be appropriately selected depending on the administration form, age, body weight, and symptoms, and is not particularly limited, but can be administered at a concentration ranging from 8.2 mM to 245.7 mM, as shown in the following examples.
以下実施例により、本発明について説明する。
[実施例１]
［がん細胞に対するエリスリトールの増殖抑制］
マウス肺がん細胞、Ｌｅｗｉｓ ｌｕｎｇ ｃａｒｃｉｎｏｍａ（ＬＬＣ）細胞（ＲＩＫＥＮ ＢＲＣ ＣＥＬＬ ＢＡＮＫ）は、９６ウェルプレート（ビーエム機器株式会社）に５×１０３細胞／ウェルにて播種し、１０％ｆｅｔａｌ ｂｏｖｉｎｅ ｓｅｒｕｍ（ＦＢＳ）（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ）を含む、９０μＬのＤＭＥＭ培地（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ）にエリスリトールを添加し、２４時間及び４８時間培養した。培地中に添加したエリスリトール（伊藤忠食糧株式会社）は、Ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒｅｄ ｓａｌｉｎｅ（ＰＢＳ）にそれぞれ０．０８２、０．８１９、２．４５７Ｍにて溶解し、０．２ｍｍ（ＡＤＶＡＮＴＥＣ）フィルターを使用して滅菌した後、培地に添加した。すなわち、最終濃度は８．２ｍＭ、８１．９ｍＭ、２４５．７ｍＭとなっている。
The present invention will now be described with reference to the following examples.
[Example 1]
[Erythritol inhibits the proliferation of cancer cells]
Mouse lung cancer cells, Lewis lung carcinoma (LLC) cells (RIKEN BRC CELL BANK) were seeded at 5 x 103 cells/well in a 96-well plate (BM Equipment Co., Ltd.), and erythritol was added to 90 μL of DMEM medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), and the cells were cultured for 24 and 48 hours. Erythritol (Itochu Foods Co., Ltd.) added to the medium was dissolved in phosphate buffered saline (PBS) at 0.082, 0.819, and 2.457 M, respectively, and sterilized using a 0.2 mm (ADVANTEC) filter, and then added to the medium. That is, the final concentrations were 8.2 mM, 81.9 mM, and 245.7 mM.
細胞増殖はｃｅｌｌ ｃｏｕｎｔｉｎｇ ｋｉｔ－８（Ｄｏｊｉｎｄｏ Ｍｏｌｅｃｕｌａｒ Ｔｅｃｈｎｏｌｏｇｉｅｓ）を用いて評価した。２４時間、４８時間培養の後、１０μＬのテトラゾリウム塩ＷＳＴ－８を添加し、３７℃で１時間静置し、４５０ｎｍの吸光度で測定した。算出された数値は、各吸光度の平均値をとった後、コントロール（ｃｏｎｔｒｏｌ）の値を１．０として相対値を算出した（図１）。 Cell proliferation was evaluated using cell counting kit-8 (Dojindo Molecular Technologies). After 24 and 48 hours of culture, 10 μL of tetrazolium salt WST-8 was added, the cells were left to stand at 37°C for 1 hour, and the absorbance at 450 nm was measured. The calculated values were calculated by taking the average of each absorbance and then calculating the relative value with the control value set at 1.0 (Figure 1).
最終濃度８．２ｍＭから２４５．７ｍＭまでの濃度でエリスリトール（図１中、ＥＲＴと記載）を培地に添加し、２４時間後、４８時間後の細胞増殖を測定した。その結果、２４時間後には８１．９ｍＭ濃度以上のエリスリトール添加で有意に（ｐ＜０．０５）、４８時間後には、８．２ｍＭでも有意に（ｐ＜０．０１）細胞増殖抑制効果が認められた。また、エリスリトールによる細胞の増殖抑制は濃度依存的であることが示された。なお、以下の図で＊はｐ＜０．０５、＊＊はｐ＜０．０１、＊＊＊はｐ＜０．００１を示す。 Erythritol (shown as ERT in Figure 1) was added to the medium at final concentrations ranging from 8.2 mM to 245.7 mM, and cell proliferation was measured after 24 and 48 hours. As a result, after 24 hours, the addition of erythritol at a concentration of 81.9 mM or higher significantly (p<0.05) inhibited cell proliferation, and after 48 hours, even at 8.2 mM (p<0.01). It was also shown that the inhibition of cell proliferation by erythritol is concentration-dependent. In the figures below, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.
細胞増殖抑制効果が生じる作用機序を検討した。がん細胞の増殖に必要なＡＴＰの供給が、エリスリトールにより抑制されている可能性があることから、細胞内ＡＴＰ濃度の解析を行った（図２）。細胞増殖抑制効果を検討したエリスリトール濃度と同濃度である８．２ｍＭから２４５．７ｍＭになるようにエリスリトールを培地に添加し、２４時間後、４８時間後のＡＴＰ量をＡＴＰ Ａｓｓａｙ Ｋｉｔ－Ｌｕｍｉｎｅｓｃｅｎｃｅ（Ｄｏｊｉｎｄｏ Ｍｏｌｅｃｕｌａｒ Ｔｅｃｈｎｏｌｏｇｉｅｓ）により解析を行った。エリスリトール添加４８時間後に、２４５．７ｍＭのエリスリトールを添加した細胞で、ＡＴＰ濃度が低下した（ｐ＜０．００１）ことから、がん細胞の増殖に必要なＡＴＰの供給が、エリスリトールにより抑制されていることが示唆される。 The mechanism of action by which the cell proliferation inhibitory effect occurs was investigated. Since it is possible that erythritol inhibits the supply of ATP necessary for the proliferation of cancer cells, the intracellular ATP concentration was analyzed (Figure 2). Erythritol was added to the medium so that the concentration was the same as the erythritol concentration used to investigate the cell proliferation inhibitory effect, from 8.2 mM to 245.7 mM, and the ATP amount after 24 hours and 48 hours was analyzed using the ATP Assay Kit-Luminescence (Dojindo Molecular Technologies). 48 hours after the addition of erythritol, the ATP concentration decreased in cells to which 245.7 mM erythritol had been added (p<0.001), suggesting that the supply of ATP necessary for the proliferation of cancer cells is inhibited by erythritol.
[実施例２]
［エリスリトールによるがん細胞増殖に関する遺伝子発現への影響］
エリスリトール添加後の遺伝子発現の変化の解析を行った。ＬＬＣ細胞の培地に、エリスリトール２４５．７ｍＭ、コントロールとしては等量のＰＢＳを添加し、４８時間後に細胞をｍｉＲＮｅａｓｙ Ｋｉｔｓ（ＱＩＡＧＥＮ）を用いて抽出し、Ｂｉｏｅｎｇｉｎｅｅｒｉｎｇ Ｌａｂ株式会社にて、ＲＮＡ－ｓｅｑを実施した。得られたデータは、ＣＬＣ Ｇｅｎｏｍｉｃｓ Ｗｏｒｋｂｅｎｃｈ（ＣＬＣ ｂｉｏ）とＧｅｎｅ Ｓｅｔ Ｅｎｒｉｃｈｍｅｎｔ Ａｎａｌｙｓｉｓ （ＧＳＥＡ）（ＵＣ Ｓａｎ Ｄｉｅｇｏ ａｎｄ Ｂｒｏａｄ Ｉｎｓｔｉｔｕｔｅ）用いて解析を実施した。
[Example 2]
[Effect of erythritol on gene expression related to cancer cell proliferation]
Analysis of changes in gene expression after the addition of erythritol was performed. 245.7 mM erythritol and an equal amount of PBS as a control were added to the LLC cell medium, and 48 hours later, the cells were extracted using miRNeasy Kits (QIAGEN), and RNA-seq was performed at Bioengineering Lab Co., Ltd. The obtained data was analyzed using CLC Genomics Workbench (CLC bio) and Gene Set Enrichment Analysis (GSEA) (UC San Diego and Broad Institute).
図３に示すように、エリスリトール添加により多数の遺伝子発現が変化していた。これら発現に変化のあった遺伝子のパスウェイ解析を行った（図４）。解糖系に関連する遺伝子ａｌｄｏａ（ａｌｄｏｌａｓｅ）とがん浸潤・転移に関連する遺伝子ｖｉｍ（ｖｉｍｅｎｔｉｎ）、ｓｎａｉｌ、ｃｄｈ（ｃａｄｈｅｒｉｎ）の発現が下がり、細胞骨格関連の遺伝子ｒｈｏｆ（Ｒａｓ Ｈｏｍｏｌｏｇ Ｆａｍｉｌｙ Ｍｅｍｂｅｒ Ｆ）、ａｃｔｎ（ａｃｔｉｎ）、ａｃｔｇ２（ａｃｔｉｎ ｇａｍｍａ ２）、ａｃｔｂ（ａｃｔｉｎ ｂｅｔａ）の発現が上昇していた。 As shown in Figure 3, the addition of erythritol changed the expression of many genes. Pathway analysis of the genes whose expression was changed was performed (Figure 4). Expression of the glycolysis-related gene aldoa (aldolase) and the cancer invasion/metastasis-related genes vim (vimentin), snail, and cdh (cadherin) decreased, while expression of the cytoskeleton-related genes rhof (Ras Homolog Family Member F), actn (actin), actg2 (actin gamma 2), and actb (actin beta) increased.
[実施例３]
［マウスモデルにおけるがん細胞の増殖抑制効果］
７週齢のＣ５７ＢＬ／６Ｊ ｍｉｃｅ（日本エスエルシー株式会社）は、１週間２３℃±２℃、１２時間ごとの明暗サイクル、実験食（ＣＥ－２ ３０ｋＧｙ照射済み、日本クレア株式会社）で１週間馴化飼育した（ｎ＝８）。その後、ＤＭＥＭで培養したＬＬＣ細胞（７継代培養）１×１０６ｃｅｌｌｓを０．２ｍＬ生理食塩水で希釈し、針付き注射筒（マイショット、２７Ｇｘ１３ｍｍ、ＮＩＰＲＯ）を用いてマウスの右鼠蹊部皮下に注射した。同時に、エリスリトールを２．５Ｍで生理食塩水に溶解し、針付き注射筒（マイショット、３０Ｇｘ１３ｍｍ、ＮＩＰＲＯ）を用いて、尾静脈より０．２ｍＬ投与した（１回投与量、６０ｍｇ、ヒト等価用量１６２．６ｍｇ／ｋｇ）。エリスリトールの尾静脈注射は、１回／１日にて実施した。実験期間中、がんの定着をゲージを使って計測し、２週間後に解剖した。癌の大きさ（ｍｍ３）は、０．４（係数）×（最小直径ｍｍｘ最大直径ｍｍ）で計算し、平均値を算出した（図５）。なお、副作用がないと考えられることから、投与量の上限は特に規定するものではない。
[Example 3]
[Cancer cell proliferation suppression effect in mouse models]
Seven-week-old C57BL/6J mice (Japan SLC Co., Ltd.) were acclimatized for one week at 23°C ± 2°C, with a 12-hour light-dark cycle, and fed with experimental food (CE-2 30 kGy irradiated, Japan CLEA Co., Ltd.) (n = 8). Then, 1 x 10 6 cells of LLC cells (7 passages) cultured in DMEM were diluted with 0.2 mL of saline and injected subcutaneously into the right groin of the mouse using a needle-equipped syringe (My Shot, 27G x 13mm, NIPRO). At the same time, erythritol was dissolved in saline at 2.5M, and 0.2 mL was administered from the tail vein using a needle-equipped syringe (My Shot, 30G x 13mm, NIPRO) (single dose, 60 mg, human equivalent dose 162.6 mg/kg). Erythritol was injected into the tail vein once per day. During the experiment, the extent of cancer colonization was measured using a gauge, and the animals were dissected after 2 weeks. The size of the cancer (mm 3 ) was calculated by 0.4 (coefficient) x (minimum diameter mm x maximum diameter mm), and the average value was calculated (Figure 5). Since there are no side effects, no upper limit for the dosage is specified.
エリスリトールの投与はがんの拡大を抑制する傾向にあり、コントロール（ＰＢＳ）と比べて、エリスリトールを投与したマウスでは、がんの拡大が遅れる傾向にあった。平均体重は投与開始時はコントロール群１８．９ｇ、エリスリトール投与群１９．１ｇ、解剖前はコントロール群１９．５ｇ、エリスリトール投与群２０．３ｇであった。実験終了時において、両群のマウス間では体重の差は認められず、重篤な副作用はないことが確認された。 Erythritol administration tended to suppress the spread of cancer, and compared to the control (PBS), cancer spread tended to be delayed in mice administered erythritol. At the start of administration, the average body weight was 18.9g in the control group and 19.1g in the erythritol-administered group, and before dissection it was 19.5g in the control group and 20.3g in the erythritol-administered group. At the end of the experiment, there was no difference in body weight between the mice in both groups, and it was confirmed that there were no serious side effects.
経口投与でも、エリスリトールの腫瘍増殖抑制効果が見られるか検討を行った。上記と同様に馴化飼育を行ったマウスに、実験食を与えるコントロール群（ＣＤ）、エリスリトールを５％配合した飼料を与えるエリスリトール群（ＥＤ）、ケストースを５％配合した飼料を与えるケストース群（ＫＤ）の３群にランダムに分け１週間飼育した（ｎ＝６）。ケストースは、構造の全く異なる糖質であり、コントロールの位置付けである。 We investigated whether erythritol's tumor growth-inhibiting effect could be seen even when administered orally. Mice were habituated in the same way as above, and then randomly divided into three groups (n=6) fed the experimental diet: a control group (CD), an erythritol group (ED), and a kestose group (KD), fed a diet containing 5% erythritol. Kestose is a carbohydrate with a completely different structure, and serves as a control.
上記と同様にして、１×１０６ｃｅｌｌｓのＬＬＣ細胞を０．２ｍＬ生理食塩水で希釈し、針付き注射筒を用いてマウスの右鼠蹊部皮下に注射した。その後、継続して各飼料で飼育しながら、がんの定着を毎日ゲージにより計測し、２週間後に解剖した。がんの大きさを測定し、腫瘍体積の平均値を算出した（図６）。コントロール群、エリスリトール投与群、ケストース投与群、いずれの群も実験期間中腫瘍体積に大きな差は認められなかった。エリスリトールは、経口投与では腫瘍の増殖に対しては効果がなく、非経口投与でのみ効果を有することが明らかとなった。 In the same manner as above, 1 x 106 LLC cells were diluted with 0.2 mL of physiological saline and injected subcutaneously into the right groin of the mice using a needle-equipped syringe. After that, the mice were continuously fed each diet, and the tumor colonization was measured daily using a gauge, and the mice were dissected after 2 weeks. The size of the tumor was measured, and the average tumor volume was calculated (Figure 6). No significant difference in tumor volume was observed during the experimental period in any of the control group, erythritol-administered group, and kestose-administered group. It was revealed that erythritol has no effect on tumor growth when administered orally, and is only effective when administered parenterally.
[実施例４]
［がん細胞の遊走能に対するエリスリトールの効果］
実施例２の結果から、エリスリトール添加により、がんの浸潤、転移に関する遺伝子発現が変化していたことから、細胞の遊走能に与える影響の解析を行った。インサート（カルチャーインサート２４ウェル用、０．４μｍ ＰＥＴ透明メンブレン、Ｆａｌｃｏｎ）に０．１％ＦＢＳを含むＤＭＥＭ培地を４００μＬ入れ、ＬＬＣ細胞を１×１０５個の細胞数でインサートに播種した。２４ウェルセルカルチャーインサートコンパニオンプレート（Ｃｏｒｎｉｎｇ）に、エリスリトールを２４５．７ｍＭになるように添加した無血清ＤＭＥＭ培地を１０００μＬ入れ、２４時間培養を行った。インサートを取り出し、アスピレーションで培地を吸引し、さらに綿棒でインサート内を拭き取った。１ｍＬの７０％エタノールが入っているウェルにインサート移し、インサート内に０．５ｍＬの７０％エタノールを加えた。３０分放置したのち、アスピレーションでインサート内の７０％エタノールを除き、１ｍＬのＰＢＳが入った新しい２４ウェルに、インサートを移した。インサート内に、ＰＢＳを０．５ｍＬ加え、インサートを洗浄した。洗浄操作を２回行ったのち、インサート内のＰＢＳをアスピレーションで除いた。
[Example 4]
[Effect of erythritol on cancer cell migration]
From the results of Example 2, the addition of erythritol changed gene expression related to cancer invasion and metastasis, so the effect on cell migration was analyzed. 400 μL of DMEM medium containing 0.1% FBS was placed in an insert (Culture insert for 24 wells, 0.4 μm PET transparent membrane, Falcon), and LLC cells were seeded on the insert at a cell count of 1 × 10 5 cells. 1000 μL of serum-free DMEM medium to which erythritol was added to make 245.7 mM was placed in a 24-well cell culture insert companion plate (Corning), and cultured for 24 hours. The insert was removed, the medium was aspirated, and the inside of the insert was wiped with a cotton swab. The insert was transferred to a well containing 1 mL of 70% ethanol, and 0.5 mL of 70% ethanol was added to the insert. After leaving it for 30 minutes, the 70% ethanol in the insert was removed by aspiration, and the insert was transferred to a new 24-well containing 1 mL of PBS. 0.5 mL of PBS was added to the insert to wash it. After washing twice, the PBS in the insert was removed by aspiration.
ギムザ（Ｇｉｅｍｓａ、Ｓｉｇｍａ－Ａｌｄｒｉｃｈ）を純水で１０倍希釈しギムザ染色液とした。１ｍＬのギムザ染色液が入った２４ウェルを用意し、インサートを置き、インサート内にさらにギムザ染色液０．５ｍＬを加えた。３０分放置し、インサート内のギムザ染色液を吸引して除いた。純水１ｍＬずつを入れた新しい２４ウェルを用意し、そこへインサートを置いた。インサート内に純水０．５ｍＬ加え、インサートを洗浄した。洗浄操作を２回行ったのち、顕微鏡下（オールインワン蛍光顕微鏡、Ｋｅｙｅｎｃｅ）で撮影を行った。画像より細胞数を数え、コントロールとの相対比較を行った（図７）。 Giemsa (Sigma-Aldrich) was diluted 10-fold with pure water to prepare the Giemsa staining solution. 24 wells containing 1 mL of Giemsa staining solution were prepared, inserts were placed in them, and 0.5 mL of Giemsa staining solution was added to the inserts. After leaving it for 30 minutes, the Giemsa staining solution in the inserts was removed by suction. 24 new wells containing 1 mL of pure water were prepared, and the inserts were placed in them. 0.5 mL of pure water was added to the inserts to wash them. After washing twice, images were taken under a microscope (all-in-one fluorescent microscope, Keyence). The number of cells was counted from the images and compared to the control (Figure 7).
エリスリトールを添加した群では、細胞の遊走能が有意に抑制されていた（ｐ＜０．０１）。この結果は、エリスリトールによって、細胞が移動する能力が抑制されること、すなわち、がん細胞の浸潤、転移が抑制される可能性を示唆している。 In the group to which erythritol was added, the migration ability of cells was significantly suppressed (p<0.01). This result suggests that erythritol may inhibit the ability of cells to migrate, that is, inhibit the invasion and metastasis of cancer cells.
[実施例５]
［エリスリトールの細胞骨格にあたえる影響］
３．５ｃｍ細胞培養ディッシュに滅菌されたカバーガラス（Ｍａｔｓｕｎａｍｉ）を置き、２４５．７ｍＭになるようにエリスリトールを添加したＤＭＥＭ培地、または、コントロールとしてＰＢＳを等量添加したＤＭＥＭ培地を入れ、２．５×１０４個のＬＬＣ細胞を播種した。４８時間後、アスピレーションで培地を除いたのち、３．７％ホルマリン２ｍＬを加えて、５分間放置し、細胞を固定した。アスピレーションでホルマリンを除き、２ｍＬ ＰＢＳ＋０．１％ ｔｒｉｔｏｎを加えた。アスピレーションでＰＢＳ＋０．１％ ｔｒｉｔｏｎを除き、２ｍＬ ＰＢＳでカバーガラスを洗浄した。アスピレーションでＰＢＳを除き、再度洗浄を行った。アクチン染色は、Ａｌｅｘａ Ｆｌｕｏｒ ４８８で標識化されたＰｈａｌｌｏｉｄｎ（１：１００、Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ）、核染色はＤＡＰＩ（１：３５０００、ａｂｃａｍ）を、１％ ｂｏｖｉｎｅ ｓｅｒｕｍ ａｌｂｍｉｎ（ＢＳＡ）で希釈し、１時間染色した。カバーガラスを０．１％ Ｔｗｅｅｎを含む１ｍＬ ＰＢＳで３回洗浄し、さらに１ｍＬ ＰＢＳで３回洗浄した。１０μＬ ｆｌｕｒｏｍｏｕｎｔ（Ｄｉａｇｎｏｓｔｉｃ Ｂｉｏｓｙｓｔｅｍｓ）でマウントし、１時間放置したのち、顕微鏡下（Ｋｅｙｅｎｃｅ）で撮影した（図８）。
[Example 5]
[Effect of erythritol on the cytoskeleton]
A sterilized cover glass (Matsunami) was placed on a 3.5 cm cell culture dish, and DMEM medium to which erythritol had been added to make it 245.7 mM, or DMEM medium to which an equal amount of PBS had been added as a control, was added, and 2.5 x 10 4 LLC cells were seeded. After 48 hours, the medium was removed by aspiration, and 2 mL of 3.7% formalin was added and left for 5 minutes to fix the cells. Formalin was removed by aspiration, and 2 mL of PBS + 0.1% triton was added. PBS + 0.1% triton was removed by aspiration, and the cover glass was washed with 2 mL of PBS. PBS was removed by aspiration, and washing was performed again. Actin staining was performed with Alexa Fluor 488-labeled Phalloidin (1:100, Thermo Fisher), and nuclear staining was performed with DAPI (1:35000, Abcam) diluted with 1% bovine serum albumin (BSA) and stained for 1 hour. The cover glass was washed three times with 1 mL PBS containing 0.1% Tween, and then washed three times with 1 mL PBS. The glass was mounted with 10 μL fluromount (Diagnostic Biosystems), left for 1 hour, and then photographed under a microscope (Keyence) (FIG. 8).
エリスリトールを添加した細胞では、細胞骨格であるアクチンが核周辺に凝集している像が見られた。一方、コントロールでは多くの細胞でそのような像は見られず、エリスリトールは細胞骨格に対しても影響を与えることが示唆された。 In cells to which erythritol was added, actin, a member of the cytoskeleton, was observed to be aggregated around the nucleus. On the other hand, in the control cells, no such images were observed, suggesting that erythritol also has an effect on the cytoskeleton.
[実施例６]
［各種糖アルコールのがん細胞への効果と抗がん剤の併用］
エリスリトールに腫瘍細胞増殖抑制効果が認められたことから、他の糖アルコールが同様の効果を有する可能性を考え、エリスリトールに加えて、キシリトール、ソルビトールについても腫瘍増殖抑制効果の解析を行った。９６ウェルマイクロタイタープレート（ビーエム機器株式会社）に、１０％ ＦＢＳを含むＤＭＥＭ培地を用い、５×１０３細胞でＬＬＣ細胞を播種し、２４時間前培養を行った。前培養ののち、エリスリトール（ＥＲＴ）、キシリトール（ＸＲＴ）、ソルビトール（ＳＲＴ）を、ＰＢＳにそれぞれ最終濃度が８．２ｍＭ、８１．９ｍＭ、２４５．７ｍＭになるように溶解し、０．２ｍｍ（ＡＤＶＡＮＴＥＣ）フィルターを使用して滅菌した後、培地に添加した。液量は９μｌ／ウェルとした。
[Example 6]
[Effects of various sugar alcohols on cancer cells and their combination with anticancer drugs]
Since erythritol was found to have a tumor cell proliferation inhibitory effect, the possibility that other sugar alcohols may have a similar effect was considered, and in addition to erythritol, xylitol and sorbitol were also analyzed for their tumor proliferation inhibitory effects. LLC cells were seeded at 5 x 103 cells in a 96-well microtiter plate (BM Equipment Co., Ltd.) using DMEM medium containing 10% FBS, and pre-cultured for 24 hours. After pre-culture, erythritol (ERT), xylitol (XRT), and sorbitol (SRT) were dissolved in PBS to final concentrations of 8.2 mM, 81.9 mM, and 245.7 mM, respectively, and sterilized using a 0.2 mm (ADVANTEC) filter, and then added to the medium. The liquid volume was 9 μl/well.
抗がん剤との併用効果を検討するために、抗がん剤としてシスプラチンをジメチルスルホシキド（Ｗａｋｏ）に溶解し、０から４０μＭの濃度で添加した。添加する液量は１μｌ／ウェルとした。評価には、ｃｅｌｌ ｃｏｕｎｔｉｎｇ ｋｉｔ－８を使用した。１０μＬの水溶性テトラゾリウム塩ＷＳＴ－８を添加し、３７℃で１時間静置し、吸光度４５０ｎｍ波長で測定した。算出された数値は、各吸光度の平均値をとった後、コントロールの値を１．０として相対値を求めた。また、２４時間、４８時間培養をしたのち、各時間で細胞数が半分になるシスプラチンの濃度（ＩＣ５０）を評価した（図９）。 To examine the combined effect with anticancer drugs, cisplatin was dissolved in dimethyl sulfoxide (Wako) as an anticancer drug and added at concentrations of 0 to 40 μM. The amount of liquid added was 1 μl/well. Cell counting kit-8 was used for the evaluation. 10 μL of water-soluble tetrazolium salt WST-8 was added, and the cells were left to stand at 37°C for 1 hour, and absorbance was measured at a wavelength of 450 nm. The calculated values were calculated by taking the average of each absorbance, and then calculating the relative value with the control value set at 1.0. In addition, after culturing for 24 and 48 hours, the concentration of cisplatin at which the number of cells was halved at each time point (IC50) was evaluated (Figure 9).
２４時間後はいずれも変化が見られなかったが、４８時間培養を行うと、エリスリトール、キシリトールは細胞増殖を抑制することが示された。エリスリトール２５４．７ｍＭを添加した場合には、シスプラチンを添加して得られたＩＣ５０は６．９μＭであり、キシリトールを８２ｍＭ添加した場合には、シスプラチンＩＣ５０は１３.８μＭ、キシリトール２４５．７ｍＭ添加ではシスプラチンＩＣ５０は、１１．５μＭだった。これらの結果から、キシリトールにも、がん細胞増殖抑制効果があるものの、キシリトールよりもエリスリトールの方が、がんの細胞の増殖抑制は顕著と言える。 After 24 hours, no changes were observed in either case, but after 48 hours of culture, it was shown that erythritol and xylitol inhibited cell proliferation. When 254.7 mM erythritol was added, the IC50 obtained by adding cisplatin was 6.9 μM, when 82 mM xylitol was added, the cisplatin IC50 was 13.8 μM, and when 245.7 mM xylitol was added, the cisplatin IC50 was 11.5 μM. From these results, it can be said that although xylitol also has an inhibitory effect on cancer cell proliferation, erythritol is more pronounced in inhibiting cancer cell proliferation than xylitol.
一方、ソルビトールは、８．２ｍＭ、８２ｍＭではコントロールと変わらず、シスプラチンの効果のみが見られた。エリスリトールとキシリトールはシスプラチンと併用することにより、８２ｍＭ、２４５．７ｍＭ濃度で相加的に細胞増殖を抑制することが示された。この結果は、シスプラチンのような細胞傷害性の抗がん剤と併用投与する場合に、他の抗がん剤が低濃度であっても効果を奏する可能性を示している。そうすると、副作用の強い細胞傷害性の抗がん剤が低濃度で作用することから、副作用を抑えて治療を行えることを示唆している。 On the other hand, sorbitol was no different from the control at 8.2 mM and 82 mM, and only the effect of cisplatin was observed. When erythritol and xylitol were used in combination with cisplatin, they were shown to additively inhibit cell proliferation at concentrations of 82 mM and 245.7 mM. These results indicate that when administered in combination with a cytotoxic anticancer drug such as cisplatin, other anticancer drugs may be effective even at low concentrations. This suggests that treatment can be carried out with reduced side effects, as cytotoxic anticancer drugs with strong side effects can act at low concentrations.
[実施例７]
［エリスリトール類縁体の細胞増殖抑制効果］
エリスリトールだけではなく、キシリトールにも細胞増殖抑制効果が認められ、ソルビトールには細胞増殖抑制効果が認められなかったことから、他の低分子の類縁化合物にがん細胞増殖抑制効果があるか検討を行った。エリスリトールの立体異性体であるＬ-トレイトール、及びエリスリトールの１位及び4位のヒドロキシ基がカルボキシル基に置き換えられたメソ酒石酸について、細胞の増殖抑制効果を検討した。検討した化合物の構造をエリスリトールや実施例６で効果が認められたキシリトールと合わせて図１０に示す。
[Example 7]
[Cytostatic effect of erythritol analogues]
Not only erythritol but also xylitol was found to have a cell proliferation inhibitory effect, but sorbitol was not found to have a cell proliferation inhibitory effect. Therefore, we investigated whether other low molecular weight analogues have a cancer cell proliferation inhibitory effect. We investigated the cell proliferation inhibitory effect of L-threitol, a stereoisomer of erythritol, and mesotartaric acid, in which the hydroxyl groups at the 1st and 4th positions of erythritol are replaced with carboxyl groups. The structures of the investigated compounds are shown in Figure 10 together with erythritol and xylitol, whose effect was confirmed in Example 6.
実施例１と同様にして、ＬＬＣ細胞を、９６ウェルプレートに５×１０３細胞／ウェルにて播種し、ＰＢＳに８．２、８１．９、２４５．７ｍＭに溶解したＬ-トレイトール（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ）、またはメソ酒石酸（Fｕｊｉｆｉｌｍ Ｗａｋｏ）を１／１０量培地に添加し、２４時間及び４８時間培養した。コントロールとしては、溶媒であるＰＢＳを等量添加した。細胞増殖をｃｅｌｌ ｃｏｕｎｔｉｎｇ ｋｉｔ－８を用いて評価した。結果を図１１に示す。 In the same manner as in Example 1, LLC cells were seeded at 5 x 103 cells/well in a 96-well plate, and L-threitol (Sigma-Aldrich) or mesotartaric acid (Fujifilm Wako) dissolved in PBS at 8.2, 81.9, or 245.7 mM was added to the medium at 1/10 volume, followed by incubation for 24 and 48 hours. As a control, an equal volume of the solvent PBS was added. Cell proliferation was evaluated using cell counting kit-8. The results are shown in FIG. 11.
メソ酒石酸は、高濃度では培地が酸性になることから、ＮａＯＨを添加して培地が中性付近となるような条件（Ｔａｒｔａｒｉｃ ａｃｉｄ＿ＮａＯＨ+）、あるいは、ｐＨを調整しない条件で（Ｔａｒｔａｒｉｃ ａｃｉｄ＿ＮａＯＨ-）解析を行った。２４５．７ｍＭのメソ酒石酸を添加した場合には、２４時間後から有意に細胞増殖抑制が認められた。ｐＨ調整を行った場合でも２４時間後から細胞増殖抑制効果が認められることから、メソ酒石酸に細胞増殖抑制効果があるものと認められる。しかしながら、エリスリトールで見られたような濃度依存的な細胞増殖抑制効果ではなかった。 Since mesotartaric acid makes the medium acidic at high concentrations, analysis was performed under conditions where NaOH was added to make the medium nearly neutral (Tartaric acid_NaOH+), or without adjusting the pH (Tartaric acid_NaOH-). When 245.7 mM mesotartaric acid was added, significant inhibition of cell proliferation was observed from 24 hours onwards. Even when the pH was adjusted, the cell proliferation inhibitory effect was observed from 24 hours onwards, indicating that mesotartaric acid has an inhibitory effect on cell proliferation. However, the inhibitory effect on cell proliferation was not concentration-dependent as seen with erythritol.
一方、Ｌ-トレイトール（Ｌ－Ｔｈｒｅｉｔｏｌ）は、２４５．７ｍＭで添加し、４８時間後であっても細胞増殖抑制効果が認められなかった。Ｌ-トレイトールは、エリスリトールと化学組成は同一であるが、細胞増殖抑制効果は認められなかったことから、立体構造が重要であると考えられる。 On the other hand, when L-threitol was added at 245.7 mM, no cell proliferation inhibitory effect was observed even after 48 hours. Although L-threitol has the same chemical composition as erythritol, the lack of cell proliferation inhibitory effect suggests that the three-dimensional structure is important.
以上、見てきたように、エリスリトール、キシリトール、メソ酒石酸には細胞増殖抑制効果があり、その中でもエリスリトールは濃度依存的に細胞増殖抑制効果があった。さらに解析を重ねたところ、エリスリトールには細胞増殖抑制効果だけではなく、遊走能を抑制する効果もあることから、がんの転移も抑制する効果があるものと認められる。エリスリトールは従来から食品や化粧品として使用されているものであり、これを有効成分とする抗がん剤は、安全性も高く、副作用もないものと考えられる。
As we have seen, erythritol, xylitol, and mesotartaric acid have cell proliferation inhibitory effects, and among these, erythritol has a concentration-dependent cell proliferation inhibitory effect. Further analysis showed that erythritol not only inhibits cell proliferation, but also inhibits migration, and is therefore considered to have an inhibitory effect on cancer metastasis. Erythritol has traditionally been used in foods and cosmetics, and anticancer drugs that contain it as an active ingredient are thought to be highly safe and have no side effects.
Claims (5)
非経口投与により投与することを特徴とする抗がん剤。 Erythritol is the active ingredient.
An anticancer agent which is administered parenterally.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008129846A1 (en) * | 2007-03-29 | 2008-10-30 | Daiichi Sankyo Company, Limited | Pharmaceutical composition |
| JP2011512397A (en) * | 2008-02-22 | 2011-04-21 | セダーマ | Moisturizing cosmetic composition comprising a combination of homarin and erythritol |
| WO2020090970A1 (en) * | 2018-10-31 | 2020-05-07 | 富士フイルム株式会社 | Pharmaceutical composition containing antitumor agent |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2008129846A1 (en) * | 2007-03-29 | 2008-10-30 | Daiichi Sankyo Company, Limited | Pharmaceutical composition |
| JP2011512397A (en) * | 2008-02-22 | 2011-04-21 | セダーマ | Moisturizing cosmetic composition comprising a combination of homarin and erythritol |
| WO2020090970A1 (en) * | 2018-10-31 | 2020-05-07 | 富士フイルム株式会社 | Pharmaceutical composition containing antitumor agent |
Non-Patent Citations (1)
| Title |
|---|
| 1 July 2021 (2021-07-01), NASSANI RAYAN, ALAMRI HASSAN, ALRFAEI BAHAUDDEEN M.: "Abstract LB183: Erythritol acts as tumor enhancer and suppressor depending on concentrations in brain tumor cell lines", XP009557963 * |
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