WO2010032704A1 - Préparation pharmaceutique comprenant un dérivé de micro-arn-143 - Google Patents

Préparation pharmaceutique comprenant un dérivé de micro-arn-143 Download PDF

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Publication number
WO2010032704A1
WO2010032704A1 PCT/JP2009/066002 JP2009066002W WO2010032704A1 WO 2010032704 A1 WO2010032704 A1 WO 2010032704A1 JP 2009066002 W JP2009066002 W JP 2009066002W WO 2010032704 A1 WO2010032704 A1 WO 2010032704A1
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WO
WIPO (PCT)
Prior art keywords
microrna
pyridine
modified
benzene
mir
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PCT/JP2009/066002
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English (en)
Japanese (ja)
Inventor
幸博 赤尾
幸夫 北出
壮一郎 竹西
朋枝 須佐
Original Assignee
財団法人岐阜県研究開発財団
国立大学法人岐阜大学
北海道システム・サイエンス株式会社
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Application filed by 財団法人岐阜県研究開発財団, 国立大学法人岐阜大学, 北海道システム・サイエンス株式会社 filed Critical 財団法人岐阜県研究開発財団
Publication of WO2010032704A1 publication Critical patent/WO2010032704A1/fr

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    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/317—Chemical structure of the backbone with an inverted bond, e.g. a cap structure
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity
    • C12N2320/51—Methods for regulating/modulating their activity modulating the chemical stability, e.g. nuclease-resistance

Definitions

  • microRNA-143 has an antitumor effect
  • Japanese Patent Application No. 2006-266918 Japanese Patent Application No. 2006-266918.
  • RNase degrading enzyme
  • the present inventors succeeded in providing a benzene / pyridine (BP) -modified microRNA-143 that has a modified sense strand that is hardly degraded by a degrading enzyme and has a higher antitumor effect than the wild type.
  • the activity of Compound 5 was 108.6 ⁇ mol / g.
  • Activity ( ⁇ mol / g) Abs, (498 nm) ⁇ Vol. (Solution) (mL) x 14.3 / Weight (support) (mg)
  • the column was washed with sterilized water to remove the salt, eluted with 3 mL of 50% CH 3 CN in H 2 O, and dried under reduced pressure.
  • 100 ⁇ L of loading solution (1 ⁇ TBE in 90% formamide) was added, and the target oligonucleotide was isolated by 20% PAGE (600 V, 20 mA).
  • the target oligonucleotide was cut out, 20 mL of 0.1 M TEAA buffer 1 mM EDTA aqueous solution was added, and the mixture was shaken for 12 hours.
  • the filtrate was passed through an equilibrated C-18 reverse phase column (Sep-Pak) and adsorbed on the column.
  • the column was washed with sterilized water to remove the salt, eluted with 3 mL of 50% CH 3 CN in H 2 O, and dried under reduced pressure.
  • the method for providing a pharmaceutical composition comprises: (A) producing BP-microRNA-143 of the present invention, (B) formulating the BP-microRNA-143 so produced with a pharmaceutically acceptable excipient.
  • administration is preferably “prophylactically effective amount” or “therapeutically effective amount” (prophylaxis can be considered a therapy, although depending on the case. This is an amount sufficient to show benefit to the individual.
  • dose and the rate and time course of administration will depend on the nature and severity of the subject being treated. Decisions regarding treatment prescriptions, such as dosages, are made at the responsibility of the general practitioner and other physicians.
  • BP-microRNA-143 or composition of the present invention can be administered alone or in combination with other treatments simultaneously or sequentially, for example, depending on the condition to be treated as described above.
  • a pharmaceutical composition using BP-microRNA-143 of the present invention comprises, in addition to the active ingredient, pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other formulations well known in the art.
  • pharmaceutically acceptable excipients can contain things. In particular, they can contain pharmaceutically acceptable excipients.
  • Such formulations should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the exact nature of the carrier or other formulation will depend on the route of administration, which may be oral administration or injection, eg, skin, subcutaneous or intravenous injection.
  • BP-microRNA-143 For intravenous, cutaneous or subcutaneous injection, or injection or application to the affected site, BP-microRNA-143 has a suitable pH, isotonicity and stability and does not contain pyrogens. It will be in the form of an aqueous solution that is acceptable. Those skilled in the art are well able to produce suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. If necessary, preservatives, stabilizers, buffers, antioxidants and / or other additives may be included.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • preservatives, stabilizers, buffers, antioxidants and / or other additives may be included.
  • the BP-microRNA-143 of the present invention can be administered locally to a specific site, or it can target specific cells or tissues, for example using intra-arterial stent-based transport It is possible to transport.
  • a targeting therapy using a targeting system such as an antibody or a cell-specific ligand can be used. Targeting is desirable to allow more compounds to enter the target cell accurately.
  • Wild-miRNA manufactured by Ambion
  • 3,4M-miR-143 BP
  • 3,4M-miR-143 TTP
  • 5% fetal calf serum at a concentration of 20 nM
  • Add to the RPMI-1640 medium and incubate at 37 ° C for 1, 2, 5, 10, 15, 30 minutes.
  • RNAse inhibitor RNase OUT 40U, manufactured by Invitrogen
  • RNase OUT 40U manufactured by Invitrogen
  • RNA degradation was generally within 5 minutes.
  • the Ct values after 30 minutes are 8.14, 5.38, and 6.51, respectively, and BP-modified synthetic 3,4M-BP-miR-143 has the largest amount of residual RNA, and in the presence of serum. It was found to be stable.
  • Antitumor effect of benzene-pyridine modified miR-143 was examined using an animal tumor model. 4 − 5 weeks old nude mice were subcutaneously inoculated with human colon cancer cell line DLD-1 2 x 10 6 cells. Tumors that appeared and the tumor diameter exceeded 1 x 0.5 cm were divided into 3 groups (1 group: 8 animals), Renilla (sea shiitake) luciferase (R), benzene-pyridine modified miR-143 Of 3,4M-BP-miR-143 and 1,2,4M-BP-miR-143 were administered locally to the tumor only 0.2 mg each time. After 4 weeks, the tumor was removed and its weight was measured.
  • the 3,4M-BP-miR-143 group of benzene-pyridine modified miR-143 significantly decreased the tumor weight compared to the control R group, and 1,2,4M- Although BP-miR-143 was effective in half of the anatomical findings, there was great variation and no significant difference. However, gross findings showed a general trend of tumor shrinkage.
  • Tumors that appeared and the tumor diameter exceeded 1 x 0.5 cm were divided into 3 groups (1 group: 5-6 mice), the control group (miR-R administration group), benzene-pyridine modified 3, 4M -A 25 ⁇ g administration group (25 ⁇ g / mouse) of BP-miR-143 and a 50 ⁇ g administration group (50 ⁇ g / mouse) were administered via the tail vein once a week for 5 consecutive weeks. Two weeks after the final administration, the tumor was excised, its weight was measured and photographed.
  • the tumor weight decreased in the low-dose group of benzene-pyridine modified 3,4M-BP-miR-143 compared to the control group.
  • the tumor weight was significantly decreased in the high-administration group of 3,4M-BP-miR-143 as compared with the control R group.
  • FIG. 6 photograph
  • a tumor reducing effect was observed depending on the dose of 3,4M-BP-miR-143.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention porte sur un dérivé de benzène-pyridine de micro-ARN-143, qui est insensible à la dégradation par une enzyme digestive (ARNase). L'invention porte également sur l'utilisation du dérivé à des fins médicales. BP-micro-ARN-143, qui a une activité supérieure à celle du micro-ARN-143 naturel et qui est insensible à la dégradation, est synthétisé par appariement d'au moins une à trois parties dans un brin sens d'une séquence (non complémentaire) ayant quatre mésappariements dans le micro-ARN-143 naturel (aux parties correspondantes dans une séquence complémentaire) et modification de l'extrémité 3' de la séquence résultante avec benzène-pyridine (BP).
PCT/JP2009/066002 2008-09-22 2009-09-14 Préparation pharmaceutique comprenant un dérivé de micro-arn-143 WO2010032704A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2008-242263 2008-09-22
JP2008242263A JP2011251912A (ja) 2008-09-22 2008-09-22 マイクロｒｎａ―１４３誘導体を含有する医薬

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WO2010032704A1 true WO2010032704A1 (fr) 2010-03-25

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140315982A1 (en) * 2011-11-02 2014-10-23 Osaka City University Double-stranded nucleic acid molecule for gene expression control
WO2018079841A1 (fr) * 2016-10-31 2018-05-03 国立大学法人岐阜大学 Molécule d'acide nucléique double brin, et son utilisation
CN109477090A (zh) * 2016-04-14 2019-03-15 e-NA生物科技公司 微小rna-143衍生物

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6216531B2 (ja) * 2013-03-29 2017-10-18 シーシーアイ株式会社 細胞増殖抑制剤およびがんの予防・治療剤
JP6899110B2 (ja) * 2017-03-21 2021-07-07 国立大学法人東海国立大学機構 細胞増殖抑制剤およびがんの予防・治療剤

Citations (5)

* Cited by examiner, † Cited by third party
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WO2006027862A1 (fr) * 2004-09-07 2006-03-16 Gifu University Analogue de nucléoside et analogue d'oligonucléotide contenant celui-ci
JP2006271387A (ja) * 2002-02-20 2006-10-12 Sirna Therapeutics Inc 短干渉核酸（ｓｉＮＡ）を用いる遺伝子発現のＲＮＡ干渉媒介性阻害
JP2007525192A (ja) * 2003-05-23 2007-09-06 サーナ・セラピューティクス・インコーポレイテッド 化学修飾した低分子干渉核酸（ｓｉＮＡ）を使用する遺伝子発現のＲＮＡ干渉仲介抑制
JP2007531520A (ja) * 2004-04-01 2007-11-08 ダーマコン， インコーポレイテッド Ｒｎａ干渉においてオフターゲット効果を減じるための修飾されたポリヌクレオチド類
JP2008086201A (ja) * 2006-09-29 2008-04-17 Gifu Prefecture Kenkyu Kaihatsu Zaidan マイクロｒｎａ生成の検出方法と癌の診断・治療およびマイクロｒｎａ生成調整剤

Patent Citations (5)

* Cited by examiner, † Cited by third party
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JP2006271387A (ja) * 2002-02-20 2006-10-12 Sirna Therapeutics Inc 短干渉核酸（ｓｉＮＡ）を用いる遺伝子発現のＲＮＡ干渉媒介性阻害
JP2007525192A (ja) * 2003-05-23 2007-09-06 サーナ・セラピューティクス・インコーポレイテッド 化学修飾した低分子干渉核酸（ｓｉＮＡ）を使用する遺伝子発現のＲＮＡ干渉仲介抑制
JP2007531520A (ja) * 2004-04-01 2007-11-08 ダーマコン， インコーポレイテッド Ｒｎａ干渉においてオフターゲット効果を減じるための修飾されたポリヌクレオチド類
WO2006027862A1 (fr) * 2004-09-07 2006-03-16 Gifu University Analogue de nucléoside et analogue d'oligonucléotide contenant celui-ci
JP2008086201A (ja) * 2006-09-29 2008-04-17 Gifu Prefecture Kenkyu Kaihatsu Zaidan マイクロｒｎａ生成の検出方法と癌の診断・治療およびマイクロｒｎａ生成調整剤

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
AKAO, Y. ET AL.: "MicroRNAs 143 and 145 are possible common onco-microRNAs in human cancers", ONCOLOGY REPORTS, vol. 16, 2006, pages 845 - 850 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140315982A1 (en) * 2011-11-02 2014-10-23 Osaka City University Double-stranded nucleic acid molecule for gene expression control
US9593331B2 (en) * 2011-11-02 2017-03-14 Osaka City University Double-stranded nucleic acid molecule for gene expression control
CN109477090A (zh) * 2016-04-14 2019-03-15 e-NA生物科技公司 微小rna-143衍生物
EP3444345A4 (fr) * 2016-04-14 2019-12-18 e-NA Biotec Inc. Dérivé d'arnmicro-143
US11041152B2 (en) 2016-04-14 2021-06-22 E-Na Biotec Inc. MicroRNA-143 derivative
CN109477090B (zh) * 2016-04-14 2022-03-22 e-NA生物科技公司 微小rna-143衍生物
WO2018079841A1 (fr) * 2016-10-31 2018-05-03 国立大学法人岐阜大学 Molécule d'acide nucléique double brin, et son utilisation
US10874688B2 (en) 2016-10-31 2020-12-29 Gifu University Double-stranded nucleic acid molecule and use thereof

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