WO2018221735A1 - Nouveaux oligonucléotides - Google Patents

Nouveaux oligonucléotides Download PDF

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WO2018221735A1
WO2018221735A1 PCT/JP2018/021246 JP2018021246W WO2018221735A1 WO 2018221735 A1 WO2018221735 A1 WO 2018221735A1 JP 2018021246 W JP2018021246 W JP 2018021246W WO 2018221735 A1 WO2018221735 A1 WO 2018221735A1
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cancer
oligonucleotide
seq
cells
pharmaceutical composition
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PCT/JP2018/021246
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Japanese (ja)
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貞佳 渡邉
博 中瀬
順一郎 中村
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富田製薬株式会社
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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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

Definitions

  • the present invention relates to an oligonucleotide having anticancer activity. Moreover, this invention relates to the pharmaceutical and anticancer agent using the said oligonucleotide.
  • an anticancer agent using a molecular target drug such as imatinib has been developed, and a high effect is recognized in cancer treatment.
  • molecular targeted drugs have side effects caused by the invasion of normal cells, and the administration can not be continued, or cancer cells acquire drug resistance, and the effects are significantly reduced.
  • cancer cells acquire drug resistance, and the effects are significantly reduced.
  • Nucleic acid drugs have the advantages of high sequence specificity and the ability to target DNA and RNA that could not be targeted by molecular targeting drugs, and thus are expected to be highly effective and safe and can be applied in various disease fields. It is being considered.
  • nucleic acid drugs that can inhibit the functions of in vivo molecules such as unique DNA, RNA, and protein existing in cancer cells are attracting attention as next-generation anticancer agents.
  • nucleic acids having anticancer activity have been intensively studied (see, for example, Patent Documents 1 to 3), but there are still few candidate products that are considered to be clinically useful as anticancer agents. is the current situation. Under such a background, development of a novel nucleic acid having an anticancer activity is demanded.
  • An object of the present invention is to provide a novel nucleic acid having anticancer activity and a medicine using the nucleic acid.
  • the present inventors have found that the oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 6 have a growth inhibitory action on cancer cells and also against normal cells. Found no effect. That is, it has been found that the oligonucleotide has an excellent cancer therapeutic effect and safety and is effective as an anticancer agent.
  • the present invention has been completed by further studies based on such knowledge.
  • Item 1 An oligonucleotide consisting of the base sequence shown in any of SEQ ID NOs: 1 to 6 below. 5'-GGTTAGGG-3 '(SEQ ID NO: 1) 5'-GGTTAGGGT-3 '(SEQ ID NO: 2) 5'-GGTTAGGGTT-3 '(SEQ ID NO: 3) 5'-GGTTAGGGTTA-3 '(SEQ ID NO: 4) 5'-GGTTAGGGTTAG-3 '(SEQ ID NO: 5) 5'-GTTAGGGT-3 '(SEQ ID NO: 6) Item 2. An anticancer agent comprising the oligonucleotide according to Item 1. Item 3.
  • a pharmaceutical composition comprising the oligonucleotide according to Item 1.
  • Item 4. The pharmaceutical composition according to Item 3, further comprising a carrier for nucleic acid delivery.
  • Item 5. The pharmaceutical composition according to Item 3 or 4, which is used for treatment of cancer or prevention of cancer metastasis.
  • Item 6. The pharmaceutical composition according to any one of Items 3 to 5, wherein the cancer is pancreatic cancer, lung cancer, osteosarcoma, or leukemia.
  • Item 7. Use of the oligonucleotide according to Item 1 for the production of an anticancer agent.
  • Item 8. Item 8. The use according to Item 7, wherein the anticancer agent is used for treatment of cancer or prevention of cancer metastasis.
  • Item 9. Item 9.
  • Item 8 wherein the cancer is pancreatic cancer, lung cancer, osteosarcoma, or leukemia.
  • Item 10 A method for treating cancer or preventing metastasis, comprising a step of administering an effective amount of the oligonucleotide according to Item 1 to a cancer patient or a person who is required to prevent cancer metastasis.
  • Item 11. Item 11. The cancer treatment or metastasis prevention method according to Item 10, wherein the cancer is pancreatic cancer, lung cancer, osteosarcoma, or leukemia.
  • the oligonucleotide of the present invention can effectively inhibit the growth of cancer cells without inhibiting the growth of normal cells, it is useful as a nucleic acid drug having an excellent cancer therapeutic effect and safety.
  • Experiment 1 it is a figure which shows the result of having evaluated the growth inhibitory effect with respect to a human pancreatic cancer cell about each oligonucleotide.
  • Experiment 2 it is a figure which shows the result of having evaluated the growth inhibitory effect with respect to a human osteosarcoma cell about each oligonucleotide.
  • Experiment 3 it is a figure which shows the result of having evaluated the growth inhibitory effect with respect to each non-small cell lung cancer cell about each oligonucleotide.
  • Experiment 4 it is a figure which shows the result of having evaluated the growth inhibitory effect with respect to a human chronic myeloid leukemia cell about each oligonucleotide.
  • Experiment 5 it is a figure which shows the result of having evaluated the growth inhibitory effect with respect to a human skin fibroblast about each oligonucleotide.
  • Experiment 6 it is a figure which shows the result of having evaluated the growth inhibitory effect with respect to a human pancreatic cancer cell about each oligonucleotide.
  • Experiment 7 it is a figure which shows the result of having evaluated the growth inhibitory effect with respect to a human pancreatic cancer cell about each oligonucleotide.
  • Experiment 8 it is a figure which shows the result of having evaluated the growth inhibitory effect with respect to a human skin fibroblast about each oligonucleotide.
  • Oligonucleotide The oligonucleotide of the present invention is characterized by comprising the base sequence shown in any of the following SEQ ID NOs: 1 to 6. 5'-GGTTAGGG-3 '(SEQ ID NO: 1) 5'-GGTTAGGGT-3 '(SEQ ID NO: 2) 5'-GGTTAGGGTT-3 '(SEQ ID NO: 3) 5'-GGTTAGGGTTA-3 '(SEQ ID NO: 4) 5'-GGTTAGGGTTAG-3 '(SEQ ID NO: 5) 5'-GTTAGGGT-3 '(SEQ ID NO: 6)
  • the base sequences shown in SEQ ID NOs: 2 to 5 are those in which T, TT, TTA, and TTAG are added to the 3 ′ end side in the base sequence shown in SEQ ID NO: 1.
  • the base sequence shown in SEQ ID NO: 6 is obtained by deleting guanine (G) at the 5 'end in the base sequence shown in SEQ ID NO: 2.
  • the oligonucleotide of the present invention specifically inhibits the growth of cancer cells without inhibiting the growth of normal cells based on having the specific base sequences shown in SEQ ID NOs: 1 to 6. Is possible.
  • oligonucleotides consisting of the base sequences shown in SEQ ID NOs: 1 to 6, preferably from the base sequences shown in SEQ ID NOs: 2, 3, 4 and 6 from the viewpoint of providing even better anticancer action and safety
  • the following oligonucleotide is mentioned.
  • the pentose linked to each base may be either D-ribose or deoxy-D-ribose, and these may be mixed. That is, the oligonucleotide of the present invention may be any of a DNA oligonucleotide, an RNA oligonucleotide, or a DNA / RNA chimeric oligonucleotide. Preferably, a DNA oligonucleotide is used. If the oligonucleotide of the present invention is an RNA oligonucleotide, T (thymine) in the nucleotide sequences shown in SEQ ID NOs: 1 to 6 is U (uracil).
  • the oligonucleotide of the present invention is a DNA / RNA chimeric oligonucleotide
  • T (thymine) is present in the RNA portion in the nucleotide sequences shown in SEQ ID NOs: 1 to 6, the T (thymine) is replaced with U (uracil).
  • the oligonucleotide of the present invention may be subjected to various modifications generally applied to nucleic acids as necessary in order to confer resistance to nucleases and the like as long as the effects of the present invention are not hindered.
  • modifications include modification of a sugar chain moiety such as 2′-O methylation; modification of a base moiety; modification of a phosphate moiety such as amination, lower alkylamination, acetylation, phosphorothioate, and the like. It is done.
  • modification of the nucleic acid can be performed according to a known technique.
  • the oligonucleotide of the present invention can be obtained by a known production method.
  • the oligonucleotide of the present invention can be obtained by chemical synthesis, or can be obtained by amplification using the complementary strand of the oligonucleotide of the present invention as a template.
  • the oligonucleotide of the present invention can effectively inhibit the growth of cancer cells without inhibiting the growth of normal cells, it can be suitably used as an anticancer agent.
  • the formulation, suitable cancer type, administration method, dose, etc. are as shown in the column of “2. Pharmaceutical composition” described later. .
  • the oligonucleotide may be formulated as a pharmaceutical composition. That is, the pharmaceutical composition of the present invention is characterized by containing the oligonucleotide.
  • the pharmaceutical composition of the present invention preferably further contains a nucleic acid delivery carrier in order to efficiently deliver the oligonucleotide into cancer cells.
  • the carrier for nucleic acid delivery is not particularly limited as long as it can be used for nucleic acid delivery and is pharmaceutically acceptable.
  • These nucleic acid delivery carriers may be used alone or in combination of two or more.
  • the usage mode of these nucleic acid delivery carriers may be appropriately set according to the type of the nucleic acid delivery carrier to be used.
  • the nucleic acid delivery carrier is bonded to the oligonucleotide by a non-covalent bond such as a hydrogen bond or a hydrophobic bond.
  • the nucleic acid delivery carrier may be used in a complex form, or a nucleic acid delivery carrier may be covalently linked to the oligonucleotide.
  • a known bifunctional linker may be used.
  • the amount of the nucleic acid delivery carrier is set to an effective amount capable of delivering the oligonucleotide into the cancer cell depending on the type, dosage form, administration method, etc. of the nucleic acid delivery carrier used. You only have to set it.
  • the dosage form of the pharmaceutical composition of the present invention is not particularly limited, and may be set as appropriate according to the administration method and the like.
  • Specific examples of the dosage form of the pharmaceutical composition of the present invention include solid preparations such as tablets, capsules, pills, powders, granules, suppositories, and patches; liquids, suspensions, emulsions, syrups, Examples thereof include liquid preparations such as injections.
  • a liquid preparation is preferable.
  • the pharmaceutical composition of the present invention is formulated by adding a pharmaceutically acceptable carrier or additive depending on the dosage form.
  • a pharmaceutically acceptable carrier or additive depending on the dosage form.
  • a pharmaceutically acceptable carrier or additive for example, in the case of a solid preparation, it can be formulated using an excipient, a binder, a disintegrant, a lubricant, a solubilizing agent, an antioxidant, an isotonic agent and the like.
  • a liquid preparation it can be formulated using physiological saline, a buffer solution, or the like.
  • the pharmaceutical composition of the present invention is used for cancer treatment or cancer metastasis prevention use.
  • the type of cancer to be treated or to prevent metastasis is not particularly limited as long as it is a cancer subject to chemotherapy, but specifically, pancreatic cancer, lung cancer, osteosarcoma, colon cancer, colon cancer , Gastric cancer, rectal cancer, liver cancer, breast cancer, bladder cancer, prostate cancer, cervical cancer, head and neck cancer, bile duct cancer, gallbladder cancer, oral cancer, melanoma, brain cancer, etc. solid cancer; leukemia, malignant lymphoma, etc. Is mentioned.
  • pancreatic cancer, lung cancer, osteosarcoma, and leukemia are preferable.
  • the administration method of the pharmaceutical composition of the present invention is not particularly limited and may be systemic administration or local administration, and may be appropriately set according to the cancer type to be applied.
  • systemic administration include intravascular (intraarterial or intravenous) administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, and inhalation administration.
  • Intravascular administration includes continuous infusion as well as intravascular injection.
  • the dose of the pharmaceutical composition of the present invention may be appropriately set within an effective range capable of exerting an anticancer effect depending on the cancer type to be applied, the degree of symptoms, the sex of the patient, age, etc.
  • the dose of the oligonucleotide per administration may be set to be about 1 ng to 300 mg / kg (body weight) and administered at a frequency of about 1 to 21 times / week.
  • composition of the present invention may contain other anticancer agents as necessary in order to exert the anticancer effect more effectively, and other anticancer agents and It may be administered in combination.
  • the type of the other anticancer agent contained or used in the pharmaceutical composition of the present invention is not particularly limited and may be appropriately set according to the cancer type to be applied. Agents, alkylating agents, microtubule agonists, anticancer antibiotics, topoisomerase inhibitors, platinum preparations and the like.
  • antimetabolites such as 5-fluorouracil, methotrexate, doxyfluridine, tegafur, 6-mercaptopurine, cytarabine, gemcitabine; alkylating agents such as clophosphamide, ifosfamide, thiotepa, carbocon, nimustine hydrochloride; docetaxel, paclitaxel, vincristine Antitubergic antibiotics such as doxorubicin hydrochloride, mitomycin, amrubicin hydrochloride, pirarubicin hydrochloride, epirubicin hydrochloride, aclarubicin hydrochloride, mitoxantrone hydrochloride, bleomycin hydrochloride, pepromycin sulfate; irinotecan, nogitecan hydrochloride; And the like, and platinum preparations such as cisplatin, oxaliplatin, carboplatin, nedaplatin and the like
  • oligonucleotide Synthesis An oligonucleotide (DNA molecule) having the base sequence shown in Table 1 was synthesized using a DNA / RNA synthesizer NTS MX-A20 (manufactured by Nippon Techno Service Co., Ltd.).
  • Test Example 1 Evaluation of growth inhibitory action on human pancreatic cancer cells
  • Human pancreatic cancer cell line (MIA PaCa-2) (purchased from JCRB Cell Bank, National Institute of Medical Science, Health and Nutrition) 100 Units / ml penicillin, 100 ⁇ g / ml streptomycin A cell solution suspended at 3.0 ⁇ 10 4 cells / ml in Eagle's minimum essential medium (E-MEM) medium containing 0.1 mM non-essential amino acids and 10% fetal bovine serum did. The cell solution was added to a 96-well plate at 100 ⁇ l / well and cultured overnight at 37 ° C. in a 5% CO 2 atmosphere.
  • E-MEM Eagle's minimum essential medium
  • the oligonucleotides of Examples 1 and 6 and Comparative Examples 1 and 2 were introduced into the cells using a transfection reagent X-treme GENE HP Transfection reagent (Roche) according to the attached protocol. Specifically, 10 ⁇ l of a mixture of the oligonucleotide and transfection reagent was added to each well to make a total volume of 110 ⁇ l. The final concentration of the oligonucleotide in the well was 5.5 ng / ⁇ l. After transfection, the cells were cultured at 37 ° C. for 48 hours. Thereafter, 10 ⁇ l of Cell Counting Kit-8 (manufactured by Dojindo) was added and incubated at 37 ° C.
  • a transfection reagent X-treme GENE HP Transfection reagent (Roche) according to the attached protocol. Specifically, 10 ⁇ l of a mixture of the oligonucleotide and transfection reagent was added to each well to make a
  • the results obtained are shown in FIG.
  • the oligonucleotide of Comparative Example 1 showed no growth inhibitory action on human pancreatic cancer cells, and the oligonucleotide of Comparative Example 2 showed growth inhibitory action on human pancreatic cancer cells.
  • the oligonucleotides of Examples 1 and 6 have a significantly higher growth inhibitory effect on human pancreatic cancer cells than Comparative Example 2 and have an excellent anticancer effect.
  • Test Example 2 Evaluation of growth inhibitory action on human osteosarcoma cells (1) Use of human osteosarcoma cell line (Saos-2) (purchased from RIKEN BioResource Center), (2) 5.0 ⁇ 10 4 Cell suspension added to a 96-well plate at 100 ⁇ l / well, (3) 100 Units / ml penicillin, 100 ⁇ g / ml streptomycin, and 10% fetal bovine serum in a medium (4) Using the oligonucleotides of Examples 1 and 6 and Comparative Example 1, the final concentration of the oligonucleotide in the well at the time of transfection was 9.1 ng / ⁇ l. Except for the above, the test was performed under the same conditions as in Test Example 1, and the cell viability when treated with each oligonucleotide was determined.
  • Test Example 3 Evaluation of Growth Inhibitory Action on Human Non-Small Cell Lung Cancer Cells (1) Use of a human non-small cell lung cancer cell line (A549) (purchased from JCRB Cell Bank, National Institute of Medical Science, Health and Nutrition) 2) The cell solution suspended at 5.0 ⁇ 10 4 cells / ml was added to the 96-well plate so as to be 100 ⁇ l / well, and (3) each of Example 1 and Comparative Example 1 The test was carried out under the same conditions as in Test Example 1 except that the final concentration of the oligonucleotide in the well at the time of transfection was changed to 9.1 ng / ⁇ l using the oligonucleotide. Cell viability was determined.
  • the oligonucleotide of Comparative Example 1 did not show a growth inhibitory action on human non-small cell lung cancer cells, but the oligonucleotide of Example 1 shows a growth inhibitory action on human non-small cell lung cancer cells. Became clear.
  • Test Example 4 Evaluation of growth inhibitory action on human chronic myeloid leukemia cells (1) Use of human chronic myeloid leukemia cell line (K562) (purchased from RIKEN BioResource Center), (2) 5.0 ⁇ A cell solution suspended to 10 4 cells / ml was added to a 96-well plate to 100 ⁇ l / well, (3) 100 Units / ml penicillin, 100 ⁇ g / ml streptomycin, and 10% bovine were added to the medium. Using RPMI-1640 containing fetal serum and (4) Using each oligonucleotide of Example 1 and Comparative Example 1, the final concentration of oligonucleotide in the well at the time of transfection was 9.1 ng / ⁇ l. Except that, the test was performed under the same conditions as in Test Example 1, and the cell viability when treated with each oligonucleotide was calculated. Asked.
  • Test Example 5 Evaluation of growth inhibitory action on human skin fibroblasts (normal cells) (1) Use of human skin fibroblast cell line (NHDF) (purchased from Takara Bio Inc.), (2) 2.5 The cell solution suspended to 10 4 cells / ml was added to a 96-well plate to 100 ⁇ l / well, (3) 100 Units / ml penicillin, 100 ⁇ g / ml streptomycin, and 10% to the medium RPMI-1640 containing fetal bovine serum was used, and (4) using each oligonucleotide of Example 1 and Comparative Example 1, the final concentration of oligonucleotide in the well at the time of transfection was 4.5 ng / ⁇ l. Except for the above, the test was conducted under the same conditions as in Test Example 1, and the cell viability when treated with each oligonucleotide was determined.
  • NHDF human skin fibroblast cell line
  • the oligonucleotide of Comparative Example 1 in which no growth inhibitory action on cancer cells was not observed in Test Examples 1 to 4 showed no growth inhibitory action on human skin fibroblasts (normal cells).
  • the oligonucleotide of Example 1 also had no growth inhibitory effect on human skin fibroblasts and had no adverse effect on normal cells. From the above results, it was revealed that the oligonucleotide of the present invention can specifically inhibit the growth of cancer cells without inhibiting the growth of normal cells.
  • Test Example 6 Evaluation of growth inhibitory action on human pancreatic cancer cells Except that the oligonucleotides of Examples 3 and 4 were used, the test was performed under the same conditions as in Test Example 1 and treated with each oligonucleotide. Cell viability was determined.
  • each of the oligonucleotides of Examples 3 and 4 was found to have a growth inhibitory action on human pancreatic cancer cells as in Example 1 and Example 6.
  • the examples were found to have excellent anticancer effects.
  • Test Example 7 Evaluation of growth inhibitory action on human pancreatic cancer cells Except that the oligonucleotides of Example 1 and Comparative Examples 3 to 6 were used, the test was performed under the same conditions as in Test Example 1 above. Cell viability upon treatment was determined.
  • Test Example 8 Evaluation of growth inhibitory action on human skin fibroblasts (normal cells) Except that each oligonucleotide of Examples 3, 4, and 6 was used, a test was performed under the same conditions as in Test Example 5, The cell viability when treated with each oligonucleotide was determined.
  • the oligonucleotides of Examples 3, 4, and 6 as in Example 1 have no growth inhibitory effect on human dermal fibroblasts (normal cells) and have an adverse effect on normal cells. It was confirmed that the
  • Test Example 9 Analysis of differential expression of protein in human pancreatic cancer cells
  • Human pancreatic cancer cell line (MIA PaCa-2) (purchased from JCRB Cell Bank, National Institute of Biomedical Innovation, Health and Nutrition) 100 Units / ml penicillin, 100 ⁇ g / ml streptomycin A cell solution suspended at 5.7 ⁇ 10 5 cells / ml in Eagle's minimum essential medium (E-MEM) medium containing 0.1 mM non-essential amino acid and 10% fetal bovine serum did. The cell solution was added to a 48-well plate at 270 ⁇ l / well and cultured overnight at 37 ° C. in a 5% CO 2 atmosphere.
  • E-MEM Eagle's minimum essential medium
  • the oligonucleotides of Example 1 and Comparative Example 3 were introduced into the cells using the transfection reagent X-treme GENE HP Transfection reagent (Roche) according to the attached protocol. Specifically, 30 ⁇ l of the mixture of oligonucleotide and transfection reagent was added to each well to make a total volume of 300 ⁇ l, and the final concentration of the oligonucleotide in the well was 3.3 ng / ⁇ l. After transfection, the cells were cultured at 37 ° C. for 24 hours. For reference, the same operation as described above was performed using only the transfection reagent.
  • the cells were washed twice with 300 ⁇ l of PBS, and the cells were lysed with 300 ⁇ l of Lysis Buffer containing 10 ⁇ g / ml Aprotinin, 10 ⁇ g / ml Leupeptin, and 10 ⁇ g / ml Pepstatin A. Centrifugation was performed at 14000 ⁇ g for 5 minutes, and the supernatant was taken to obtain a cell extract. Using this cell extract, a membrane was prepared using the Proteome Profiler Apoptosis Array Kit (R & D SYSTEMS) according to the attached protocol, and measured using GE ImageQuant LAS4000mini (GE Life Sciences). An expression difference analysis was performed.
  • the oligonucleotide of Example 1 by introducing the oligonucleotide of Example 1 into the cells, active oxygen is decomposed by Catalase as in Comparative Example 3, but hem-2 is decomposed by HO-2, thereby divalent iron. It is possible that free oxygen was released and active oxygen was produced. Therefore, the oligonucleotide of Example 1 induces an increase in active oxygen in cancer cells and induces cell death by making it impossible to maintain the balance between the expression level of Catalase and the amount of active oxygen. It was suggested to inhibit proliferation.
  • Test Example 10 Evaluation of antitumor effect on human pancreatic cancer cells by animal experiment 2.1 ⁇ 10 6 human pancreatic cancer cell lines (MIA PaCa-2) were administered subcutaneously to the right foot of a mouse, and the tumor size was 200 mm. After reaching 3 , the drugs of Example 1 and Comparative Example 1 were prepared using the transfection reagent in vivo jetPEI transfection reagent (manufactured by Polyplus) according to the attached protocol, and directly administered to the tumor. Specifically, 100 ⁇ l of a mixture of the oligonucleotide and the transfection reagent was administered per mouse.
  • the dosage per oligonucleotide was set to 0.5 mg / kg and 2.0 mg / kg in Example 1 and 2.0 mg / kg in Comparative Example 1.
  • drug administration was carried out for a total of 7 cycles (7 weeks), taking 3 doses every other day, followed by 2 days of withdrawal as 1 cycle (1 week).
  • PBS group a group to which only the same amount of PBS was added
  • Vehicle group a group to which only the same amount of transfection reagent was administered
  • existing anticancer agents gemcitabine hydrochloride was administered (the same number of cycles as above, with a dose of 80 mg / kg / dose twice a week (dose once every 3 to 4 days) as one cycle (one week)).
  • a positive control group was also tested in the same manner. In the test, 4 mice were used for each group, and a multiple comparison test was performed using the Bonferroni method.
  • Example 1 The obtained results are shown in FIG. In the PBS group, the Vehicle group, and Comparative Example 1, it was confirmed that the tumor continued to grow with the passage of the test days. On the other hand, in Example 1, it was confirmed that the growth of the tumor was suppressed, and particularly that the growth of the tumor was suppressed even when the dose was small (0.5 mg / kg). Furthermore, in Example 1, when there is a large dose (2.0 mg / kg), not only is there a significant difference from the Vehicle group, but it is equivalent to the existing anticancer drug gemcitabine hydrochloride (positive control group). There was an anti-tumor effect. Therefore, it was confirmed that Example 1 has an excellent antitumor effect.

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Abstract

L'objet de la présente invention est de fournir de nouveaux acides nucléiques présentant une activité antitumorale. Les oligonucléotides comprenant des séquences de bases telles que représentées par les SEQ ID No : 1 à 6 présentent une action inhibitrice de la croissance contre les cellules cancéreuses, mais n'affectent pas les cellules normales, et sont ainsi utiles en tant qu'agents anticancéreux.
PCT/JP2018/021246 2017-06-02 2018-06-01 Nouveaux oligonucléotides WO2018221735A1 (fr)

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Citations (3)

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WO1999003507A1 (fr) * 1997-07-17 1999-01-28 Board Of Regents Of The University Of Nebraska Composes mimetiques de telomeres et leurs procedes d'utilisation
JP2003513887A (ja) * 1999-09-10 2003-04-15 ジェロン・コーポレーション オリゴヌクレオチドｎ３’→ｐ５’チオホスホルアミデート：それらの合成および使用
JP2005522520A (ja) * 2002-04-12 2005-07-28 トラスティーズ オブ ボストン ユニバーシティ オリゴヌクレオチドの使用によって細胞増殖を阻止する方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999003507A1 (fr) * 1997-07-17 1999-01-28 Board Of Regents Of The University Of Nebraska Composes mimetiques de telomeres et leurs procedes d'utilisation
JP2003513887A (ja) * 1999-09-10 2003-04-15 ジェロン・コーポレーション オリゴヌクレオチドｎ３’→ｐ５’チオホスホルアミデート：それらの合成および使用
JP2005522520A (ja) * 2002-04-12 2005-07-28 トラスティーズ オブ ボストン ユニバーシティ オリゴヌクレオチドの使用によって細胞増殖を阻止する方法

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TOMOHIRO, T. ET AL.: "Total analysis and purification of cellular proteins binding to cisplatin-damaged DNA using submicron beads", BIOCONJUGATE CHEM., vol. 13, no. 2, 2002, pages 163 - 166, XP055562410 *

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