WO2015020194A1 - Antisense oligonucleotide against acsl1 - Google Patents

Antisense oligonucleotide against acsl1 Download PDF

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WO2015020194A1
WO2015020194A1 PCT/JP2014/071011 JP2014071011W WO2015020194A1 WO 2015020194 A1 WO2015020194 A1 WO 2015020194A1 JP 2014071011 W JP2014071011 W JP 2014071011W WO 2015020194 A1 WO2015020194 A1 WO 2015020194A1
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antisense oligonucleotide
sequence
seq
acsl1
substituted
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PCT/JP2014/071011
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French (fr)
Japanese (ja)
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聡 小比賀
剛史 山本
真一郎 堀
孝博 藤原
隆史 小林
武史 粕谷
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国立大学法人大阪大学
塩野義製薬株式会社
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Publication of WO2015020194A1 publication Critical patent/WO2015020194A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y602/00—Ligases forming carbon-sulfur bonds (6.2)
    • C12Y602/01—Acid-Thiol Ligases (6.2.1)
    • 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
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • 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
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • 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
    • C12N15/1137—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 against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • 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
    • C12N2310/315—Phosphorothioates
    • 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
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA

Definitions

  • the present invention relates to an antisense oligonucleotide to ACSL1 (acyl-CoA synthetase long-chain family member 1). More specifically, the present invention relates to an antisense oligonucleotide against ACSL1, which is useful as a preventive or therapeutic agent (including a pharmaceutical composition for weight management) of obesity or diabetes (particularly type II diabetes).
  • Obesity is a systemic increase in adipose tissue and occurs when the amount of energy consumed over a long period is greater than the amount of energy consumed. Obesity can be classified into visceral fat type obesity and subcutaneous fat type obesity. Visceral fat-type obesity is obesity in which the accumulation of intra-abdominal fat existing around the omentum and mesentery increases, and diabetes (particularly type II diabetes with insulin resistance), arteriosclerosis, liver disease, heart This is one of the main causes of illness and is a major problem in modern society.
  • Diabetes mellitus is a disease accompanied by a persistent hyperglycemic state, and is considered to result from the action of many environmental factors and genetic factors.
  • the main regulator of blood glucose in the body is insulin
  • hyperglycemia is caused by insulin deficiency or excessive factors that inhibit its action (eg, genetic predisposition, lack of exercise, obesity, stress, etc.) .
  • type I diabetes caused by a decrease in pancreatic insulin secretion function due to autoimmune diseases and the like and a decrease in pancreatic insulin secretion function due to pancreatic exhaustion associated with continuous high insulin secretion II Classified as type 2 diabetes. More than 95% of Japanese diabetic patients are said to have type II diabetes, and today, the increase in the number of patients accompanying lifestyle changes is a problem.
  • Enzymes belonging to the acyl-CoA synthase family are enzymes that convert long-chain fatty acids into acyl CoA. Since acyl-CoA is a substrate in intracellular lipid synthesis and fatty acid degradation or elongation reactions, ACS plays a central role in intracellular lipid metabolism as well as intracellular signaling by lipids. ACS is also involved in the uptake of extra fatty acids (see Non-Patent Document 1).
  • ACSL1 GenBank: NM_001995
  • ACSL4 and ACSL5 are mainly expressed in the liver
  • ACSL3 and ACSL6 are mainly expressed in the brain.
  • Triacsin C is known as an inhibitor of ACS, and this compound has been reported to inhibit 1, 3 and 4 of 5 isozymes (see Non-Patent Document 3).
  • this compound in addition to this compound, in addition to inhibiting TG accumulation in HuH7 cells, which are human hepatoma cell lines (see Non-Patent Document 4), and diacylglycerol, cholesterol esters, phospholipids in CCD cells, which are human normal dermal fibroblasts. It has been reported to inhibit synthesis (see Non-Patent Document 5).
  • ACSL1 has been reported to be associated with various cancers (see, for example, Patent Documents 1 to 3), and as a biomarker for cirrhosis, liver fibrosis (see Patent Document 4), and bronchial asthma (see Patent Document 5). It has also been reported.
  • Patent Document 6 discloses that suppression of hepatic ACSL1 expression by siRNA suppresses increase in body weight and lowers blood glucose level, and ACSL1 expression inhibitor is used for obesity or type II diabetes. It has been suggested that it can be used for treatment or prevention.
  • Patent Document 7 discloses an antisense compound targeting ACSL1.
  • 3615 antisense oligonucleotides are described, but only predictive values regarding affinity with a target region are described, and data regarding suppression of ACSL1 expression is not described.
  • An object of the present invention is to provide a novel antisense oligonucleotide having excellent ACSL1 expression suppression activity.
  • Patent Document 7 describes an antisense oligonucleotide for ACSL1 of 3,615, but only describes a predicted value for affinity with a target region, and does not describe data regarding suppression of ACSL1 expression.
  • antisense having high affinity does not necessarily have a high target gene expression-inhibiting action (Antisense Drug Technology Principles, Strategies, and Applications, CRC Press; 2nd edition, 2007-120, p. , FIG. 5.3a). Therefore, from the description in Patent Document 7, it cannot be predicted which region of ACSL1 mRNA is useful as the target region of the antisense oligonucleotide.
  • the antisense oligonucleotide of the present invention binds to a specific target region found by the present inventors and exhibits excellent ACSL1 expression suppression activity.
  • the present inventors have found that the antisense oligonucleotide of the present invention does not have an expression suppressing action on other isozymes (ACSL3, ACSL4 and ACSL5) and has an ACSL1-specific expression suppressing action.
  • an antisense oligonucleotide with high specificity for a target sequence is useful as a medicine.
  • the present inventors measured aspartate aminotransferase (AST) and alanine aminotransferase (ALT) upon administration of the antisense oligonucleotide of the present invention, and confirmed that no liver toxicity was observed. Therefore, the antisense oligonucleotide of the present invention has low toxicity and is sufficiently safe for use as a medicine.
  • the antisense oligonucleotide of the present invention has good metabolic stability and water solubility, and is sufficiently safe for use as a medicine.
  • the present invention relates to the following.
  • An antisense oligonucleotide comprising a sequence capable of hybridizing to a sequence consisting of positions 832 to 853, 3635 to 3654, or 3659 to 3676 of SEQ ID NO: 1 under stringent conditions.
  • the antisense oligonucleotide according to (1) which suppresses the expression of ACSL1.
  • the cross-linking structure is 4′-CH 2 —O-2 ′ or 4′-CO—NR 1 -2 ′ (R 1 is a hydrogen atom or alkyl), Sense oligonucleotide.
  • Sense oligonucleotide (6) The antisense oligonucleotide according to any one of (1) to (5), wherein one or more bonds between nucleosides are phosphorothioate bonds.
  • a pharmaceutical composition comprising the antisense oligonucleotide according to any one of (1) to (8).
  • the pharmaceutical composition according to (10), wherein the disease is obesity or type II diabetes.
  • (1-1) the sequence described in any one of SEQ ID NOs: 321 to 362, or An antisense oligonucleotide comprising a sequence according to any one of SEQ ID NOs: 321 to 362, wherein one or several bases are deleted, substituted or inserted.
  • (1-2) The antisense oligonucleotide according to (1-1), which suppresses the expression of ACSL1.
  • (1-3) The antisense oligonucleotide according to (1-1) or (1-2), which has a length of 13 to 19 bases.
  • the crosslinked structure is 4′-CH 2 —O-2 ′ or 4′-CO—NR 1 -2 ′ (R 1 is a hydrogen atom or alkyl)
  • R 1 is a hydrogen atom or alkyl
  • a pharmaceutical composition comprising the antisense oligonucleotide according to any one of (1-1) to (1-7).
  • the pharmaceutical composition according to (1-8) which is used for prevention or treatment of a disease associated with ACSL1.
  • the pharmaceutical composition according to (1-9) wherein the disease is obesity or type II diabetes.
  • SEQ ID NO: 90, 133, 164, 194, 221, 234, 244, 254, 267, 273, 280, 284, 285, 286, 293 or 302, or SEQ ID NO: 90, 133, 164, 194, 221, 234, 244, 254, 267, 273, 280, 284, 285, 286, 293 or 302 lacks one or several bases.
  • An antisense oligonucleotide comprising a missing, substituted or inserted sequence.
  • the antisense oligonucleotide according to (2-1) which suppresses the expression of Acsl1.
  • the crosslinked structure is 4′-CH 2 —O-2 ′ or 4′-CO—NR 1 -2 ′ (R 1 is a hydrogen atom or alkyl)
  • R 1 is a hydrogen atom or alkyl
  • (2-7) The antisense oligonucleotide according to any one of (2-1) to (2-6), wherein one or more bonds between nucleosides are phosphorothioate bonds.
  • a pharmaceutical composition comprising the antisense oligonucleotide according to any one of (2-1) to (2-7).
  • the crosslinked structure is 4′-CH 2 —O-2 ′ or 4′-CO—NR 1 -2 ′ (R 1 is a hydrogen atom or alkyl)
  • R 1 is a hydrogen atom or alkyl
  • a pharmaceutical composition comprising the antisense oligonucleotide according to any one of (3-1) to (3-7).
  • the antisense oligonucleotide of the present invention exhibits excellent ACSL1 expression-suppressing activity, and is associated with drugs, particularly diseases involving ACSL1, such as obesity, obesity-related diseases, diabetes (particularly type II diabetes), syndrome X, cardiovascular disorders Or it is very useful as a medicine (including a medicine for weight management) for preventing or treating cancer (breast cancer, colon cancer, colon cancer, ovarian cancer, lung cancer, etc.).
  • an “antisense oligonucleotide” is an oligonucleotide complementary to a target gene mRNA, mRNA precursor or ncRNA, and is composed of single-stranded DNA, RNA and / or analogs thereof. The The function of mRNA, mRNA precursor or ncRNA is suppressed by forming a double strand with the mRNA, mRNA precursor or ncRNA targeted by the antisense oligonucleotide.
  • Antisense oligonucleotides include not only those that are completely complementary to the target mRNA, mRNA precursor or ncRNA, but also those that can hybridize to the mRNA, mRNA precursor or ncRNA under stringent conditions. Or, there are cases where several mismatches exist.
  • An oligonucleotide means a nucleotide in which a plurality of identical or different nucleosides are bound.
  • Nucleoside means a compound in which a nucleobase and a sugar form an N-glycoside bond.
  • Nucleotide means a compound in which a phosphate group is bonded to a nucleoside sugar.
  • An analog of DNA or RNA means a molecule having a structure similar to DNA or RNA.
  • peptide nucleic acid (PNA) etc. are mentioned.
  • ncRNA non-coding RNA
  • PNA peptide nucleic acid
  • ncRNA non-coding RNA
  • One or several mismatches means 1 to 5, preferably 1 to 3, more preferably 1 or 2 mismatches.
  • ACSL1 is mentioned as a target gene of the antisense oligonucleotide of the present invention.
  • human ACSL1, mouse Acs11 and the like can be mentioned, but not limited thereto.
  • ACSL1 is a known protein.
  • the DNA sequence of human ACSL1 (GenBank: NM — 001995) is described in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence is described in SEQ ID NO: 2.
  • the DNA sequence of mouse Acsl1 (GenBank: NM — 007981) is described in SEQ ID NO: 3 and the amino acid sequence is described in SEQ ID NO: 4.
  • “ACSL1” in the present invention is not limited to these sequences, and as long as the function of the protein of SEQ ID NO: 2 or 4 is maintained, the number of amino acid and DNA mutations and mutation sites are not limited. .
  • the length of the antisense oligonucleotide of the present invention is 6 to 50 bases.
  • an antisense oligonucleotide comprising a sequence hybridizable to a sequence consisting of positions 832 to 853, 3635 to 3654, or 3659 to 3676 of SEQ ID NO: 1 under stringent conditions; or (b ) An antisense oligonucleotide containing a sequence that can hybridize to the sequence consisting of positions 883 to 899, 3718 to 3737, or 3741 to 3759 of SEQ ID NO: 3 under stringent conditions.
  • Each of the target regions in (a) is a region associated with the knockdown activity of the antisense oligonucleotide, particularly in human ACSL1 mRNA.
  • Each of the target regions in (b) is a region associated with the knockdown activity of the antisense oligonucleotide, particularly in the mouse Acsl1 mRNA. Any sequence capable of hybridizing to the target region under stringent conditions is included in the antisense oligonucleotide of the present invention regardless of the length or the presence or absence of nucleotide modification.
  • ACSL1 expression inhibitory activity can be measured by a known method. For example, it can be measured by the method described in Examples 4 to 7 described later.
  • antisense oligonucleotide of the present invention specifically, (A) a sequence consisting of positions 832 to 853, 3635 to 3654 or 3659 to 3676 of SEQ ID NO: 1, or (b) 883 to 899, 3718 to 3737 or 3741 of SEQ ID NO: 3.
  • An antisense oligonucleotide having at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more homology with a complementary sequence of the sequence consisting of ⁇ 3759 positions.
  • the homology is indicated in the score by using a search program BLAST using an algorithm developed by Altschul et al. (The Journal of Molecular Biology, 215, 403-410 (1990)), for example. .
  • “Stringent conditions” means that only antisense oligonucleotides (A) a sequence consisting of positions 832 to 853, 3635 to 3654 or 3659 to 3676 of SEQ ID NO: 1, or (b) 883 to 899, 3718 to 3737 or 3741 of SEQ ID NO: 3.
  • a base sequence that does not form a hybrid (so-called non-specific hybrid) with the specific sequence means that it forms a hybrid (so-called specific hybrid) with the sequence consisting of ⁇ 3759 and does not have an equivalent function.
  • Those skilled in the art can easily select such conditions by changing the temperature during the hybridization reaction and washing, the salt concentration of the hybridization reaction solution and the washing solution, and the like. Specifically, it is 42 ° C.
  • Examples of the stringent conditions of the present invention include, but are not limited to, the conditions in which the cells are hybridized with and washed with 0.5 ⁇ SSC at 42 ° C.
  • a hybridization method a well-known and commonly used method in this field, for example, a Southern blot hybridization method or the like can be used. Specifically, Molecular Cloning: A Laboratory Manual, Second Edition (1989) (Cold Spring Harbor Laboratory Press), Current Protocol in Amplification in Coal (1994) (Ten-Proc. It can be carried out according to the method described in Second Edition (1995) (Oxford University Press) and the like.
  • antisense oligonucleotide of the present invention for example, (A) the sequence described in any of SEQ ID NOS: 310 to 320, or A sequence in which one or several bases are deleted, substituted or inserted in the sequence shown in any of SEQ ID NOS: 310 to 320; (B) the sequence described in any one of SEQ ID NOs: 321 to 362, or A sequence in which one or several bases are deleted, substituted or inserted in the sequence of any one of SEQ ID NOs: 321 to 362, (C) SEQ ID NOs: 363, 365-369, 375-378, 380-389, 392-394, 398, 400-403, 405, 408-413, 415-418, 423-425, 428, 430-433, 436 437, 440, 443-447, 450, 452, 453, 456-458, 462, 464-469, 473-475, 477, 478, 480-482, 484-486, 489, 491-498,
  • each of these has human ACSL1 expression inhibitory activity.
  • the sequence is included, it is included in the antisense oligonucleotide of the present invention regardless of the length or nucleotide modification.
  • the antisense oligonucleotide of the present invention for example, The sequence according to any of SEQ ID NOs: 90, 133, 164, 194, 221, 234, 244, 254, 267, 273, 280, 284, 285, 286, 293 or 302, or SEQ ID NO: 90, 133, 164, 194, 221, 234, 244, 254, 267, 273, 280, 284, 285, 286, 293 or 302 lacks one or several bases.
  • Antisense oligonucleotides containing missing, substituted or inserted sequences are included. More preferably, The sequence according to any of SEQ ID NOS: 133, 164, 244, 280, 285 or 286, or Examples include antisense oligonucleotides containing sequences in which one or several bases have been deleted, substituted or inserted in the sequence set forth in any of SEQ ID NOs: 133, 164, 244, 280, 285 or 286. Each of these has mouse Acsl1 expression inhibitory activity. As long as the sequence is included, it is included in the antisense oligonucleotide of the present invention regardless of the length or nucleotide modification.
  • antisense oligonucleotide of the present invention specifically, (A-2) an antisense nucleotide of any of AON numbers 344 to 399, or An antisense nucleotide of any one of AON Nos. 344 to 399, wherein one or several bases are deleted, substituted or inserted; (B-2) an antisense nucleotide of any of AON numbers 400 to 469, 486 to 510, or An antisense nucleotide in which one or several bases are deleted, substituted or inserted in any one of the antisense nucleotides of AON Nos.
  • an antisense nucleotide of any of AON numbers 470-485 or Examples include antisense nucleotides in which one or several bases have been deleted, substituted, or inserted in any one of AON numbers 470 to 485.
  • the antisense nucleotide of any one of AON numbers 471, 472, 476, 480, 482 or 483, or Examples include antisense nucleotides in which one or several bases are deleted, substituted or inserted in any one of the antisense nucleotides of AON Nos. 471, 472, 476, 480, 482 or 483. Each of these has mouse Acsl1 expression inhibitory activity. As long as it consists of the same sequence, it is included in the antisense oligonucleotide of the present invention regardless of whether or not the nucleotide is modified.
  • one or several bases means 1 to 5, preferably 1 to 3, more preferably 1 or 2 bases.
  • the antisense oligonucleotide of the present invention includes any deletion, substitution, insertion or addition as long as it has an action of suppressing the expression of the target gene (for example, ACSL1).
  • the antisense oligonucleotide of the present invention has not only ACSL1 expression-suppressing activity but also usefulness as a medicine, and has any or all of the following excellent features.
  • a) The inhibitory effect on CYP enzymes (for example, CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, etc.) is weak.
  • the nucleotide may be modified.
  • Antisense oligonucleotides with appropriate modifications have any or all of the following characteristics compared to unmodified antisense oligonucleotides. a) High affinity with the target gene. b) High resistance to nucleases. c) Improved pharmacokinetics. d) Increases organizational transferability. Therefore, the modified antisense oligonucleotide is less likely to be degraded in vivo than the unmodified antisense oligonucleotide, and can inhibit the expression of the target gene more stably.
  • nucleotide modification known in the art can be used for the antisense oligonucleotide of the present invention.
  • Known modifications of nucleotides include phosphate modification, nucleobase modification, and sugar modification.
  • phosphoric acid modifications include phosphodiester bonds, S-oligos (phosphorothioates), D-oligos (phosphodiesters), M-oligos (methyl phosphonates), boranophosphates, etc. possessed by natural nucleic acids.
  • nucleobase modification include 5-methylcytosine, 5-hydroxymethylcytosine, 5-propynylcytosine and the like.
  • sugar modification examples include 2′-O—CH 2 —CH 2 —O—CH 3 (2′MOE), LNA (Locked Nucleic Acid) represented by Formula (a) described in Example 1 described later, Examples thereof include 2′-OMe represented by the formula (b), amide BNA (Bridged nucleic acid, AmNA) represented by the formula (c), and the like.
  • Nucleotide modification and modification methods known in the art are also disclosed in, for example, the following patent documents.
  • S-oligo phosphorothioate
  • S-oligo type phosphorothioate type
  • LNA Locked Nucleic Acid
  • a 2'-hydroxyl group is attached from the 4 'carbon atom of the sugar ring of the nucleotide via an appropriate bridge to form a bicyclic sugar moiety.
  • a preferred bond is a methylene (—CH 2 —) group that bridges the 2 ′ oxygen atom and the 4 ′ carbon atom.
  • Amide BNA Bridged nucleic acid, AmNA
  • An amide bond is formed between the 2 ′ amino group of the sugar ring of the nucleotide and the carbonyl group extended from 4 ′ to form a bicyclic sugar moiety.
  • Specific examples of amide BNA and the preparation method thereof are described in WO 2011/052436.
  • the antisense oligonucleotide of the present invention is preferably a gapmer.
  • a gapmer includes a central region (“gap”) and regions on both sides of the central region, wings (“5 ′ wing” on the 5 ′ side or “3 ′ wing” on the 3 ′ side), and each wing has at least It means an oligonucleotide containing one modified nucleotide.
  • the modification of the modified nucleotide may be any of phosphate modification, base modification, and sugar modification.
  • the type, number, and position of the modification in one wing may be the same as or different from the type, number, and position of the modification in the other wing.
  • the antisense of the present invention contains “one or more sugar-modified nucleosides having a bridging structure between the 4′-position and the 2′-position”. Gapmer is preferable.
  • “5 ′ wing” and cocoon / or “3 ′ wing” include one or more sugar-modified nucleosides having a cross-linked structure between the 4′-position and the 2′-position, preferably 1-5, more preferably 2- 3 contained.
  • phosphate modification, base modification, and other sugar modifications may be included.
  • the “sugar-modified nucleoside having a crosslinking structure between the 4′-position and the 2′-position” is a known nucleoside containing a modification having a crosslinking structure between the 4′-position and the 2′-position of the sugar, Either is acceptable. Furthermore, base modification and other sugar modifications may be included.
  • crosslinked structure examples include the following. 4 ′-(CR 2 R 3 ) m —O-2 ′, 4 ′-(CR 2 R 3 ) m —NR 1 —O-2 ′, 4 ′-(CR 2 R 3 ) n —CO—NR 1 -2 'or 4'-(CR 2 R 3 ) n -CO-NR 1 -X-2 ', here, X is an oxygen atom, a sulfur atom, amino or CR 2 R 3 , R 1 is a hydrogen atom, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aromatic carbocyclic group, substituted or unsubstituted non-aromatic carbocycle Group, substituted or unsubstituted aromatic heterocyclic group, substituted or unsubstituted non-aromatic heterocyclic group, substituted or unsubsti
  • R 1 is preferably a hydrogen atom, alkyl, alkenyl, alkynyl, aromatic carbocyclic group, non-aromatic carbocyclic group, aromatic heterocyclic group, non-aromatic heterocyclic group, aromatic carbocycle It is alkyl, non-aromatic carbocyclic alkyl, aromatic heterocyclic alkyl or non-aromatic heterocyclic alkyl, and may have one or more arbitrary substituents selected from ⁇ group.
  • the ⁇ group is a hydroxyl group, alkyl, alkyloxy, mercapto, alkylthio, amino, alkylamino or halogen.
  • R 2 and R 3 are preferably a hydrogen atom.
  • m is preferably 1 or 2.
  • n is preferably 0 or 1.
  • the cross-linked structure is preferably 4 ′-(CR 2 R 3 ) m —O-2 ′ or 4 ′-(CR 2 R 3 ) n —CO—NR 1 ⁇ 2 ′.
  • R 1 is a hydrogen atom, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl or substituted or unsubstituted alkynyl
  • Each R 2 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl
  • Each R 3 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl
  • m is 1 to 3
  • n is 0-3.
  • the cross-linked structure is particularly preferably 4′-CH 2 —O-2 ′ or 4′-CO—NR 1 -2 ′ (R 1 is a hydrogen atom or alkyl).
  • Halogen includes fluorine atom, chlorine atom, bromine atom and iodine atom. In particular, a fluorine atom and a chlorine atom are preferable.
  • Alkyl includes straight or branched hydrocarbon groups having 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, and still more preferably 1 to 4 carbon atoms. To do. For example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl , Isooctyl, n-nonyl, n-decyl and the like.
  • alkyl examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and n-pentyl. Further preferred examples include methyl, ethyl, n-propyl, isopropyl and tert-butyl.
  • Alkenyl has 2 to 15 carbon atoms, preferably 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and further preferably 2 to 4 carbon atoms, having one or more double bonds at any position. These linear or branched hydrocarbon groups are included.
  • alkenyl include vinyl, allyl, propenyl, isopropenyl, butenyl, isobutenyl, prenyl, butadienyl, pentenyl, isopentenyl, pentadienyl, hexenyl, isohexenyl, hexadienyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, decenyl, tridecenyl, decenyl Etc.
  • alkenyl include vinyl, allyl, propenyl, isopropenyl and butenyl.
  • Alkynyl has 2 to 10 carbon atoms, preferably 2 to 8 carbon atoms, more preferably 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms, having one or more triple bonds at any position. Includes straight chain or branched hydrocarbon groups. Examples include ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl and the like. These may further have a double bond at an arbitrary position. Preferred embodiments of “alkynyl” include ethynyl, propynyl, butynyl and pentynyl.
  • “Aromatic carbocyclic group” means a monocyclic or bicyclic or more cyclic aromatic hydrocarbon group. For example, phenyl, naphthyl, anthryl, phenanthryl and the like can be mentioned. A preferred embodiment of the “aromatic carbocyclic group” includes phenyl.
  • non-aromatic carbocyclic group means a cyclic saturated hydrocarbon group or a cyclic non-aromatic unsaturated hydrocarbon group having one or more rings.
  • the non-aromatic carbocyclic group having two or more rings includes a monocyclic ring or a non-aromatic carbocyclic group having two or more rings condensed with the ring in the above “aromatic carbocyclic group”.
  • the “non-aromatic carbocyclic group” includes a group which forms a bridge or a spiro ring as described below.
  • the monocyclic non-aromatic carbocyclic group preferably has 3 to 16 carbon atoms, more preferably 3 to 12 carbon atoms, and still more preferably 4 to 8 carbon atoms.
  • Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclohexadienyl, and the like.
  • Examples of the two or more non-aromatic carbocyclic groups include indanyl, indenyl, acenaphthyl, tetrahydronaphthyl, fluorenyl and the like.
  • “Aromatic heterocyclic group” means a monocyclic or bicyclic or more aromatic cyclic group having one or more heteroatoms arbitrarily selected from O, S and N in the ring. To do.
  • the aromatic heterocyclic group having two or more rings includes those obtained by condensing a ring in the above “aromatic carbocyclic group” to a monocyclic or two or more aromatic heterocyclic group.
  • the monocyclic aromatic heterocyclic group is preferably 5 to 8 members, more preferably 5 or 6 members.
  • Examples include pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazolyl, triazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, oxazolyl, oxadiazolyl, isothiazolyl, thiazolyl, thiadiazolyl and the like.
  • bicyclic aromatic heterocyclic group examples include indolyl, isoindolyl, indazolyl, indolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, naphthyridinyl, quinoxalinyl, purinyl, pteridinyl, benzimidazolyl, benzisoxazolyl, benzisoxazolyl, Oxazolyl, benzoxiadiazolyl, benzisothiazolyl, benzothiazolyl, benzothiadiazolyl, benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl, imidazopyridyl, triazolopyridyl, imidazothiazolyl, pyrazinopyr Dazinyl, oxazolopyridyl, thiazolopyridyl and the like can be mentioned
  • aromatic heterocyclic group having 3 or more rings examples include carbazolyl, acridinyl, xanthenyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, dibenzofuryl and the like.
  • non-aromatic heterocyclic group is a monocyclic or bicyclic or more cyclic non-aromatic cyclic group having one or more of the same or different heteroatoms arbitrarily selected from O, S and N in the ring Means group.
  • the non-aromatic heterocyclic group having two or more rings includes the above-mentioned “aromatic carbocyclic group”, “non-aromatic carbocyclic group”, and monocyclic or two or more non-aromatic heterocyclic groups, and Also included are those in which each ring in the “aromatic heterocyclic group” is condensed.
  • non-aromatic heterocyclic group also includes a group that forms a bridge or a spiro ring as described below.
  • the monocyclic non-aromatic heterocyclic group is preferably 3 to 8 members, more preferably 5 or 6 members.
  • Alkyloxy means a group in which the above “alkyl” is bonded to an oxygen atom. Examples thereof include methoxy, ethoxy, n-propyloxy, isopropyloxy, n-butyloxy, tert-butyloxy, isobutyloxy, sec-butyloxy, pentyloxy, isopentyloxy, hexyloxy and the like. Preferable embodiments of “alkyloxy” include methoxy, ethoxy, n-propyloxy, isopropyloxy, tert-butyloxy.
  • Alkylamino includes monoalkylamino and dialkylamino.
  • “Monoalkylamino” means a group in which the above “alkyl” is replaced with one hydrogen atom bonded to the nitrogen atom of the amino group.
  • methylamino, ethylamino, isopropylamino and the like can be mentioned.
  • methylamino and ethylamino are used.
  • “Dialkylamino” means a group in which the above “alkyl” is replaced with two hydrogen atoms bonded to the nitrogen atom of the amino group. Two alkyl groups may be the same or different.
  • Examples include dimethylamino, diethylamino, N, N-diisopropylamino, N-methyl-N-ethylamino, N-isopropyl-N-ethylamino and the like. Preferable examples include dimethylamino and diethylamino.
  • Alkylthio means a group in which the above “alkyl” is bonded to a sulfur atom.
  • substituents include the following substituents.
  • the carbon atom at any position may be bonded to one or more groups selected from the following substituents.
  • Substituents halogen, hydroxy, carboxy, amino, imino, hydroxyamino, hydroxyimino, formyl, formyloxy, carbamoyl, sulfamoyl, sulfanyl, sulfino, sulfo, thioformyl, thiocarboxy, dithiocarboxy, thiocarbamoyl, cyano, nitro, nitroso , Azide, hydrazino, ureido, amidino, guanidino, trialkylsilyl, alkyloxy, alkenyloxy, alkynyloxy, haloalkyloxy, alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, monoalkylamino, dialkylamino, alkylsulfonyl, alkenylsulfonyl, alkynyl Sulfonyl, monoalkyl
  • An atom at any position on the ring may be bonded to one or more groups selected from the following substituents.
  • substituents halogen, hydroxy, carboxy, amino, imino, hydroxyamino, hydroxyimino, formyl, formyloxy, carbamoyl, sulfamoyl, sulfanyl, sulfino, sulfo, thioformyl, thiocarboxy, dithiocarboxy, thiocarbamoyl, cyano, nitro, nitroso , Azide, hydrazino, ureido, amidino, guanidino, trialkylsilyl, alkyl, alkenyl, alkynyl, haloalkyl, alkyloxy, alkenyloxy, alkynyloxy, haloalkyloxy, alkyloxyalkyl, alkylcarbonyl, alkylcarbonyl, alkyl,
  • alkyl part of “aromatic carbocyclic alkyl”, “non-aromatic carbocyclic alkyl”, “aromatic heterocyclic alkyl”, and “non-aromatic heterocyclic alkyl” is the same as the above “alkyl”.
  • “Aromatic carbocyclic alkyl” means an alkyl substituted with one or more of the above “aromatic carbocyclic groups”. For example, benzyl, phenethyl, phenylpropynyl, benzhydryl, trityl, naphthylmethyl, groups shown below Etc.
  • aromatic carbocyclic alkyl Preferable embodiments of “aromatic carbocyclic alkyl” include benzyl, phenethyl and benzhydryl.
  • Non-aromatic carbocyclic alkyl means alkyl substituted with one or more of the above “non-aromatic carbocyclic groups”.
  • the “non-aromatic carbocyclic alkyl” also includes “non-aromatic carbocyclic alkyl” in which the alkyl moiety is substituted with the above “aromatic carbocyclic group”. For example, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, groups shown below Etc.
  • “Aromatic heterocyclic alkyl” means alkyl substituted with one or more of the above “aromatic heterocyclic groups”. “Aromatic heterocyclic alkyl” also includes “aromatic heterocyclic alkyl” in which the alkyl moiety is substituted with the above “aromatic carbocyclic group” and / or “non-aromatic carbocyclic group”. .
  • pyridylmethyl furanylmethyl, imidazolylmethyl, indolylmethyl, benzothiophenylmethyl, oxazolylmethyl, isoxazolylmethyl, thiazolylmethyl, isothiazolylmethyl, pyrazolylmethyl, isopyrazolylmethyl, pyrrolidinylmethyl, benz Oxazolylmethyl, group shown below Etc.
  • Non-aromatic heterocyclic alkyl means an alkyl substituted with one or more of the above “non-aromatic heterocyclic groups”.
  • the alkyl portion is substituted with the above “aromatic carbocyclic group”, “non-aromatic carbocyclic group” and / or “aromatic heterocyclic group”.
  • non-aromatic heterocyclic alkyl For example, tetrahydropyranylmethyl, morpholinylethyl, piperidinylmethyl, piperazinylmethyl, groups shown below Etc.
  • the antisense oligonucleotide of the present invention (or a modified product thereof) can be synthesized by a conventional method.
  • it can be easily synthesized with a commercially available automatic nucleic acid synthesizer (for example, manufactured by AppliedBiosystems, manufactured by Dainippon Seiki Co., Ltd.).
  • a commercially available automatic nucleic acid synthesizer for example, manufactured by AppliedBiosystems, manufactured by Dainippon Seiki Co., Ltd.
  • Examples of the synthesis method include a solid phase synthesis method using phosphoramidite and a solid phase synthesis method using hydrogen phosphonate. For example, it is disclosed in Tetrahedron Letters 22, 1859-1862 (1981), International Publication No. 2011/052436, and the like.
  • the antisense oligonucleotides of the present invention can be any pharmaceutical that can provide (directly or indirectly) a biologically active metabolite or residue thereof when administered to an animal, including a human. Acceptable salts, esters, or salts of such esters, or any other equivalent. That is, it includes prodrugs and pharmaceutically acceptable salts of the antisense oligonucleotides of the invention, pharmaceutically acceptable salts of the prodrugs, and other biological equivalents.
  • prodrug is an inactive or less active form that is converted into an active form (ie, drug) in the body or cell by the action and / or state of an endogenous enzyme or other chemical. Is a derivative.
  • the prodrug of the antisense oligonucleotide of the present invention can be prepared according to the methods described in WO 93/24510, WO 94/26764 and the like.
  • “Pharmaceutically acceptable salt” refers to a physiologically and pharmaceutically acceptable salt of an antisense oligonucleotide of the invention, ie, retains the desired biological activity of the antisense oligonucleotide; It refers to salts that do not give unwanted toxicological effects.
  • Examples of pharmaceutically acceptable salts include alkali metals (eg, lithium, sodium, potassium, etc.), alkaline earth metals (eg, calcium, barium, etc.), magnesium, transition metals (eg, zinc, iron, etc.), Ammonia, organic bases (eg trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, meglumine, diethanolamine, ethylenediamine, pyridine, picoline, quinoline etc.) and salts with amino acids, or inorganic acids (eg hydrochloric acid, Sulfuric acid, nitric acid, carbonic acid, hydrobromic acid, phosphoric acid, hydroiodic acid, etc.) and organic acids (eg formic acid, acetic acid, propionic acid, trifluoroacetic acid, citric acid, lactic acid, tartaric acid, oxalic acid, maleic acid) , Fumaric acid, mandelic acid, gluta Acid, mal
  • the present invention also includes a pharmaceutical composition containing the antisense oligonucleotide of the present invention.
  • a pharmaceutical composition containing the antisense oligonucleotide of the present invention As the administration method and preparation of the pharmaceutical composition of the present invention, any administration method and preparation known in the art can be used.
  • Antisense oligonucleotide administration methods and preparations are also disclosed in, for example, the following documents. International Publication No. 2004/016749, International Publication No. 2005/083124, International Publication No. 2007/143315, International Publication No. 2009/071680, International Publication No. 2013/0889283, and the like.
  • Examples of the pharmaceutical composition containing the antisense oligonucleotide of the present invention include a double-stranded nucleic acid containing a nucleic acid complementary to the antisense oligonucleotide of the present invention.
  • the double-stranded nucleic acid enables an antisense oligonucleotide to be delivered to a target site with high specificity and efficiently, and enables the expression of a target gene to be effectively suppressed by the antisense oligonucleotide.
  • Specific examples of the double-stranded nucleic acid and a method for preparing the double-stranded nucleic acid are described in International Publication No. 2013/0889283.
  • the pharmaceutical composition of the present invention can be administered by various methods depending on whether local or systemic treatment is desired or on the region to be treated.
  • the administration method may be, for example, topical (including eye drops, intravaginal, rectal, intranasal, transdermal), oral, or parenteral.
  • Parenteral administration includes intravenous injection or infusion, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration by inhalation or inhalation, intradural administration, intraventricular administration, and the like.
  • compositions for oral administration include powders, granules, suspensions or solutions dissolved in water or non-aqueous media, capsules, powders, tablets and the like.
  • compositions for parenteral, subdural space, or intraventricular administration include sterile aqueous solutions containing buffers, diluents and other suitable additives.
  • the pharmaceutical composition of the present invention comprises various pharmaceutical additives such as excipients, binders, wetting agents, disintegrants, lubricants, diluents and the like suitable for the dosage form in the effective amount of the antisense oligonucleotide of the present invention.
  • the agent can be obtained by mixing as necessary. In the case of an injection, it may be sterilized with an appropriate carrier to form a preparation.
  • Excipients include lactose, sucrose, glucose, starch, calcium carbonate or crystalline cellulose.
  • binder include methyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, gelatin, and polyvinyl pyrrolidone.
  • disintegrant include carboxymethyl cellulose, sodium carboxymethyl cellulose, starch, sodium alginate, agar powder, or sodium lauryl sulfate.
  • lubricant include talc, magnesium stearate or macrogol. As a suppository base, cocoa butter, macrogol, methylcellulose, or the like can be used.
  • solubilizers when preparing as liquid or emulsion or suspension injections, commonly used solubilizers, suspending agents, emulsifiers, stabilizers, preservatives, isotonic agents, etc. are added as appropriate. You may do it. In the case of oral administration, flavoring agents, fragrances and the like may be added.
  • the optimal dosing schedule can be calculated from measurements of drug accumulation in the body. Persons of ordinary skill in the art can determine optimum dosages, dosing methodologies and repetition rates. The optimal dose will vary depending on the relative potency of the individual antisense oligonucleotides, but can generally be calculated based on the IC50 or EC50 in in vitro and in vivo animal experiments.
  • an antisense oligonucleotide derived from the antisense oligonucleotide sequence and chemical structure
  • an effective dose derived experimentally
  • IC50 for example, mg / kg
  • ACSL1 related diseases include obesity (including weight management in obesity), obesity related diseases, diabetes (especially type II diabetes), syndrome X, cardiovascular disorder or cancer (breast cancer, colon cancer, colon cancer, Ovarian cancer, lung cancer, etc.).
  • An “obesity related disease” is a disease associated with, caused by, or caused by obesity.
  • Examples of obesity-related diseases include bulimia, hypertension, impaired glucose tolerance, diabetes, metabolic syndrome, lipid metabolism disorder, arteriosclerosis, hyperuricemia, gout, fatty liver, proteinuria, obese nephropathy, endometrium Cancer, breast cancer, prostate cancer, colon cancer, osteoarthritis, low back pain, lumbar spondylosis, obstructive sleep apnea syndrome, coronary artery disease (myocardial infarction, coronary heart disease such as angina pectoris), cerebral infarction, cerebral thrombus Disease, transient cerebral ischemic attack, menstrual abnormalities, Prader-Willi syndrome, Frehrich syndrome, Pickwick syndrome and the like.
  • the pharmaceutical composition of the present invention is also useful for reducing the risk of secondary outcomes of obesity, such as reducing the risk of left ventricular hypertrophy.
  • the pharmaceutical composition of the present invention can be used particularly for the prevention or treatment of obesity or type II diabetes.
  • the pharmaceutical composition of the present invention When the pharmaceutical composition of the present invention is used for the prevention or treatment of obesity, other one or more known anti-obesity drugs (pharmaceutical compositions containing a compound having an anti-obesity action, obesity and obesity) It can also be used in combination with a drug that can be used for weight management and the like. Moreover, the administration therapy of the pharmaceutical composition of the present invention can also be used in combination with known diet therapy, drug therapy, exercise and the like.
  • a method for the prevention or treatment of obesity or obesity-related diseases or weight management in obesity comprising administering a known anti-obesity drug in combination with the pharmaceutical composition of the present invention.
  • a method for the prevention or treatment of obesity or obesity-related diseases or weight management in obesity comprising administering a known anti-obesity drug to a patient undergoing prevention or treatment by administration of the pharmaceutical composition of the present invention.
  • Known anti-obesity drugs include compounds having an appetite suppressing action (selective serotonin reuptake inhibitors, etc.), compounds having an action to suppress digestion and absorption of nutrients ( ⁇ -glucosidase inhibitors; SGLT-2 inhibitors, etc.), fat Compounds having absorption-inhibiting action (lipase inhibitor; bile acid adsorption resin, etc.), 5HT transporter inhibitor, NE transporter inhibitor, CB-1 antagonist / inverse agonist, ghrelin antagonist, H3 antagonist / inverse agonist, MCH R1 antagonist , MCH R2 agonist / antagonist, NPY Y1 receptor antagonist, NPY Y2 receptor agonist, NPY Y4 receptor agonist, NPY Y5 receptor antagonist, mGluR5 antagonist, leptin, leptin Gonist, leptin derivative, opioid antagonist, orexin antagonist, BRS3 agonist, CCK-A agonist, CNTF, CNTF agonist,
  • the pharmaceutical composition of the present invention when used for the prevention or treatment of type II diabetes, it can also be used in combination with one or more other known type II diabetes therapeutic agents.
  • insulin secretagogues for example, sulfonylurea (SU) drugs
  • fast-acting insulin secretagogues for example, phenylalanine derivative drugs
  • glucose absorption inhibitors for example, ⁇ -glucosidase inhibitors) ( ⁇ GI drug)
  • insulin sensitizers for example, biguanide drugs (BG drugs), thiazolidine derivatives (TZD drugs)
  • insulin preparations for example, peptidyl peptidase IV (DPP-IV) inhibitors, GLP-1 receptors
  • DPP-IV peptidyl peptidase IV
  • GLP-1 receptors examples include pharmaceutical compositions containing agonists, type 1 sodium-dependent glucose transporter (SGLT1) inhibitors, type 2 sodium-dependent glucose transporter (SGLT2) inhibitors, and the like.
  • the timing of administration is not limited, and it may be administered simultaneously to the administration subject or may be administered with a time difference.
  • the pharmaceutical composition of the present invention and the other drug may be administered as a plurality of preparations containing each active ingredient, or may be administered as a single preparation containing both active ingredients.
  • LNA represented by the formula (a) or 2′-OMe represented by the formula (b) was outsourced to Gene Design Co., Ltd.
  • Base is 5-methylcytosine (C), thymine (T), adenine (A) or guanine (G).
  • Base is cytosine (C), uracil (U), adenine (A) or guanine (G), and Me is methyl.
  • the oligonucleotide containing amide BNA (AmNA) represented by the formula (c) was synthesized with reference to the method described in WO 2011/052436.
  • Base is 5-methylcytosine (C), thymine (T), adenine (A) or guanine (G), and Me is methyl.
  • a 10mer to 19mer oligonucleotide containing an LNA represented by the formula (a), 2′-OMe represented by the formula (b) or an amide BNA (AmNA) represented by the formula (c) is used in an automatic nucleic acid synthesizer (nS -8 type, manufactured by Dainippon Seiki Co., Ltd.) and was synthesized on a 0.2 ⁇ mol scale.
  • nS -8 type manufactured by Dainippon Seiki Co., Ltd.
  • Example 2 Human or mouse antisense oligonucleotide sequence
  • Antisense oligonucleotide (AON) is targeted to human ACSL1 (GenBank: NM_001995, SEQ ID NO: 1) or mouse Acsl1 (GenBank: NM_007981, SEQ ID NO: 3) Designed.
  • the oligonucleotide sequences are shown in Tables 1-26. In the target site column in the table, m represents the target site of the mouse sequence, and h represents the target site of the human sequence. In the sequences of Tables 1 to 20, capital letters represent LNAs represented by formula (a). Lower case letters represent DNA.
  • uppercase letters represent LNAs represented by formula (a).
  • Lower case letters represent DNA.
  • a e and G e represent 2′-OMe represented by the formula (b).
  • Example 3 In Vitro Model Cell Culture Cells were cultured in the appropriate media described below and maintained at 37 ° C., 95-98% humidity and 5% CO 2 .
  • HLE Human hepatoma-derived cell line HLE was cultured in DMEM Low Glucose (Sigma) + 10% fetal bovine serum (FBS) + Penicillin (100 units / mL) + Streptomycin (100 ug / mL).
  • Hepa1c1c7 The mouse liver cancer-derived cell line Hepa1c1c7 was cultured in ⁇ -MEM (Gibco) + 10% FBS + Antibiotic Anticolytic Solution (10 mL / L).
  • Example 4 Evaluation of antisense oligonucleotides against ACSL1 (1) Evaluation by introduction experiment using cell introduction reagent Mouse Hepa1c1c7 cells using antisense oligonucleotides designed and manufactured as described in Examples 1 and 2 Alternatively, knockdown experiments were performed with human HLE cells. Introduction into mouse Hepa1c1c7 cells was performed using Lipofectamine RNAiMAX reagent (invitrogen). The final concentration of antisense oligonucleotide was added to the cell culture solution to 20 nM.
  • the primer sequence used to measure the expression level of mouse Acsl1 is: Fw primer: AGGTGCTTCAGCCCACATC (SEQ ID NO: 507); Rv primer: AAAGTCCAACAGCCATCGCTTC (SEQ ID NO: 508) Use The primer sequence used to measure the expression level of mouse Gapdh is: Fw primer: TGTGTCCGTCGTGGATCTGA (SEQ ID NO: 509); Rv primer: TTGCTGTTTGAAGTCGCAGAG (SEQ ID NO: 510) was used. Human HLE cells were introduced using Lipofectamine LTX reagent (invitrogen). The final concentration of antisense oligonucleotide was added to the cell culture solution to 20 nM.
  • the primer sequence used to measure the expression level of human ACSL1 is Fw primer: GCAGCGGGCATCATCAGAAAC (SEQ ID NO: 517); Rv primer: TGTCACATCATAGCCCGACTC (SEQ ID NO: 518) Use The primer sequence used to measure the expression level of human GAPDH is: Fw primer: GCACCGTCAAGGCTGGAAC (SEQ ID NO: 519); Rv primer: TGGTGAAGACGCCAGTGGA (SEQ ID NO: 520) was used. The results are shown in Tables 30 to 32.
  • Table 30 shows the amount of Acsl1 mRNA decreased in mouse Hepa1c1c7 cells normalized with Gapdh as knockdown efficiency for antisense oligonucleotides introduced into cells using Lipofectamine RNAiMAX reagent.
  • Table 31 or Table 32 shows the amount of decrease in ACSL1 mRNA in human HLE cells normalized with GAPDH as knockdown efficiency for antisense oligonucleotides introduced into cells using Lipofectamine LTX reagent. .
  • the antisense oligonucleotide of the present invention showed high knockdown activity against mouse Acsl1 or human ACSL1 compared to other antisense oligonucleotides.
  • the primer sequence used to measure the expression level of mouse Acsl3 is: Fw primer: GCAACAACGCAGCGATTCA (SEQ ID NO: 511); Rv primer: AGCAAACTAATGGTGCCTCCACTC (SEQ ID NO: 512) was used.
  • the primer sequence used to measure the expression level of mouse Acsl4 is: Fw primer: GCCATGGAAGCTGAAATACGAAAG (SEQ ID NO: 513); Rv primer: GAAGGGCATCTGTTACAAAACCAGTC (SEQ ID NO: 514) was used.
  • the primer sequence used to measure the expression level of mouse Acsl5 is: Fw primer: CATTCGGCGGGACAGTTTG (SEQ ID NO: 515); Rv primer: ATCCCATTGCCAGCCCCTGAAG (SEQ ID NO: 516) was used.
  • the primer sequence used to measure the expression level of human ACSL3 is: Fw primer: ATACGGGCTCACTGAATCTGCTG (SEQ ID NO: 521); Rv primer: AGCAAACTAATGGTGCCTCCACTC (SEQ ID NO: 522) was used.
  • the primer sequence used to measure the expression level of human ACSL4 is Fw primer: GAATGGATGATGTCAGCACAGA (SEQ ID NO: 523); Rv primer: CCTCAGATTCATTTCCCCCATGAAC (SEQ ID NO: 524) was used.
  • the primer sequence used to measure the expression level of human ACSL5 is Fw primer: GGAACTCTGAAGATCATCGACCGTA (SEQ ID NO: 525); Rv primer: CGTTGTCAGGAACCACCACTCCTA (SEQ ID NO: 526) was used.
  • mice were collected with Fastlane (QIAGEN) or CellAmp RNA Prep Kit (Takara) 120 hours after transfection, and quantitative analysis was performed by One Step SYBR PrimeScript PLUS RT-PCR Kit (Takara). went. GAPDH was used as an endogenous control.
  • the primer sequences used for measuring the expression levels of mouse Acsl1 and mouse Gapdh were those shown in (1) above.
  • the primer sequence used to measure the expression level of human ACSL1 is Fw primer: GCAGCGGGCATCATCAGAAAC (SEQ ID NO: 517); Rv primer: TGTCACATCATAGCCCGACTC (SEQ ID NO: 518) Use The primer sequence used to measure the expression level of human GAPDH is: Fw primer: GCACCGTCAAGGCTGGAAC (SEQ ID NO: 519); Rv primer: TGGTGAAGACGCCAGTGGA (SEQ ID NO: 520) was used.
  • Results are shown in Tables 35-46.
  • the amount of decrease in ACSL1 mRNA normalized by GAPDH is shown as the ratio of untreated cells as knockdown efficiency.
  • the antisense oligonucleotide of the present invention showed high knockdown activity against mouse Acsl1 and / or human ACSL1 in Hepa1c1c7 cells and / or HLE cells.
  • the results are shown in Tables 47-52.
  • the ratio of the decrease in mRNA of ACSL3, ACSL4, or ACSL5 normalized by GAPDH to the untreated cells is shown as the knockdown efficiency.
  • N in the table.
  • D Means that the amount of mRNA decrease in ACSL3, ACSL4 or ACSL5 is below the detection limit, or the amount of mRNA is increased, and ACSL3, ACSL4 or ACSL5 is not suppressed.
  • the antisense oligonucleotide of the present invention does not knock down other ACSL families (ACSL3, ACSL4, ACSL5) more than 50% even when added to the cell culture medium at 5 ⁇ M, and shows high specificity to ACSL1. It was.
  • the sequences in which the mouse and human target sites are described in the target site column are designed as sequences for the same part of the human and mouse ACSL1 gene, respectively. Even if the sequences are the same in human and mouse, they do not always show specific and high knockdown activity. Therefore, the antisense oligonucleotide of the present invention showing both specific and high knockdown activity is Medicinal very useful.
  • Example 5 Evaluation of in vivo activity at the time of a single administration of an antisense oligonucleotide C57BL / 6J (male, 10 weeks old, CLEA, Japan) was dissolved in physiological saline (Otsuka raw food injection, Otsuka Pharmaceutical Factory). About 0.2 mL of the sense oligonucleotide solution was subcutaneously administered so that the dose per mouse individual was 10 mg / kg or 20 mg / kg. About 0.5 mL of whole blood and liver tissue were collected under somnenopentyl anesthesia 3 days, 7 days, and 14 days after administration. RNA extraction from the liver was performed according to the manufacturer's recommended protocol using RNeasy 96 Universal Tissue Kit (Qiagen).
  • RNA 1000 ng was reverse-transcribed according to a standard protocol using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (manufactured by Life Science) to obtain cDNA. Quantitative PCR was performed using SYBR Premix Ex Taq II (manufactured by Takara Bio Inc.). GAPDH was used as an endogenous control, and the same primers as in Example 4 were used. The results are shown in Tables 53 and 54. In each table, the ratio of the ACSL1 mRNA amount normalized by GAPDH to the physiological saline administration group is shown as the knockdown efficiency. As a result, it was confirmed that the antisense oligonucleotide of the present invention showed a concentration-dependent knockdown activity even in vivo.
  • Example 6 Evaluation of in vivo activity at the time of repeated administration of antisense oligo C57BL / 6J (male 7-week-old, Claire, Japan) is given a high fat fat diet (60% kcal fat: manufactured by TestDiet) for 4 weeks.
  • a high fat fat diet (60% kcal fat: manufactured by TestDiet) for 4 weeks.
  • DIO diet-induced obese mice.
  • About 0.2 mL of the antisense oligonucleotide solution of the present invention dissolved in physiological saline (Otsuka raw food injection, Otsuka Pharmaceutical Factory) was subcutaneously administered once weekly to DIO mice. The dose was 5 mg / kg / week, 10 mg / kg / week, and 20 mg / kg / week, and the dose was administered once a week for 2 weeks.
  • the antisense oligonucleotide of the present invention suppresses the increase in body weight and suppresses blood glucose level by suppressing the expression of ACSL1 in the liver. Therefore, it does not exhibit liver toxicity due to its action in the liver. Useful.
  • Example 7 Evaluation of in vivo activity at the time of frequent administration of antisense oligo C57BL / 6J (male 7 weeks old, Claire, Japan) is given 4 weeks and a high fat fat diet (60% kcal fat: manufactured by TestDiet) for 4 weeks Thus, diet-induced obese (DIO) mice were produced. About 0.2 mL of the antisense oligonucleotide solution of the present invention dissolved in physiological saline (Otsuka raw food injection, Otsuka Pharmaceutical Factory) was subcutaneously administered once weekly to DIO mice. The dose was 10 mg / kg / week and 20 mg / kg week per individual mouse, and the administration was continued for 4 weeks.
  • physiological saline Otsuka raw food injection, Otsuka Pharmaceutical Factory
  • Example 8 Effect of inhibiting weight gain upon repeated administration of antisense oligo C57BL / 6J (male 7-week-old, Claire, Japan) is given a high fat fat diet (60% kcal fat: manufactured by TestDiet) for 4 weeks.
  • a high fat fat diet (60% kcal fat: manufactured by TestDiet) mice.
  • DIO diet-induced obese mice.
  • About 0.2 mL of the antisense oligonucleotide solution of the present invention dissolved in physiological saline (Otsuka raw food injection, Otsuka Pharmaceutical Factory) once a week. It was administered subcutaneously. The dose was adjusted to 10 mg / kg / week and 20 mg / kg per mouse individual, and the administration was continued for 4 weeks.
  • the weight transition is shown in FIG. 2, and the change in food intake is shown in FIG.
  • none of the antisense oligonucleotide administration groups of the present invention showed a significant change in food intake, but none of the groups administered with the antisense oligonucleotide of the present invention. Inhibition of weight gain was confirmed. From this result, the anti-obesity effect was confirmed by suppressing the ACSL1 expression in the liver with an antisense oligo.
  • Example 9 Liver toxicity evaluation at the time of single administration of antisense oligo The toxicity in mouse liver was evaluated for the antisense oligonucleotide of the present invention. Specifically, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) contained in plasma were measured as liver toxicity markers.
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • AST and ALT in the obtained plasma were measured using a transaminase C2 kit Wako (manufactured by Wako Pure Chemical Industries, Ltd.) according to the attached manual. The results are shown in Tables 57 to 59. As a result, no significant increase in AST and ALT was observed in the blood of mice administered with the antisense oligonucleotide of the present invention as compared to the physiological saline-administered group, and the antisense oligonucleotide of the present invention showed liver toxicity. It was confirmed that there was no.
  • the antisense oligonucleotide of the present invention suppresses the increase in body weight and suppresses blood glucose level by suppressing the expression of ACSL1 in the liver. Therefore, it does not exhibit liver toxicity due to its action in the liver. Useful.
  • Example 10 Evaluation of in vivo activity of antisense oligo-human sequences
  • sequences that are 100% identical to cynomolgus monkeys are administered to cynomolgus monkeys and analyzed for the expression level of ACSL1 in the liver
  • a human sequence antisense oligo solution dissolved in physiological saline is repeatedly administered, and then the liver is excised and examined for changes in mRNA and protein expression.
  • the antisense oligonucleotide of the present invention exhibits ACSL1 expression inhibitory activity. Therefore, the compound of the present invention can prevent or prevent obesity, obesity-related diseases, diabetes (particularly type II diabetes), syndrome X, cardiovascular disorder or cancer (breast cancer, colon cancer, colon cancer, ovarian cancer, lung cancer, etc.) or the like. It is very useful as a medicine for treatment (including a medicine for weight management).

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Abstract

　It has been discovered that an antisense oligonucleotide including a sequence that can hybridize with sequences comprising positions 832-853, 3635-3654, or 3659-3676 of SEQ ID No. 1 exhibits superior inhibitory activity on the expression of ACSL1. This pharmaceutical composition contains the antisense oligonucleotide against ACSL1 as the effective ingredient thereof, and is useful for the prevention and treatment of obesity and type-II diabetes.

Description

ＡＣＳＬ１に対するアンチセンスオリゴヌクレオチドAntisense oligonucleotide to ACSL1
　本発明は、ＡＣＳＬ１（acyl-CoA synthetase long-chain family member 1）に対するアンチセンスオリゴヌクレオチドに関する。より詳細には、肥満症又は糖尿病（特に、ＩＩ型糖尿病）の予防又は治療薬（体重管理用医薬組成物も含む）として有用なＡＣＳＬ１に対するアンチセンスオリゴヌクレオチドに関する。 The present invention relates to an antisense oligonucleotide to ACSL1 (acyl-CoA synthetase long-chain family member 1). More specifically, the present invention relates to an antisense oligonucleotide against ACSL1, which is useful as a preventive or therapeutic agent (including a pharmaceutical composition for weight management) of obesity or diabetes (particularly type II diabetes).
　肥満は脂肪組織が全身的に増加した状態をいい、長期間にわたり摂取するエネルギー量が消費するエネルギー量よりも多いときに起きる。肥満は、内臓脂肪型肥満と皮下脂肪型肥満とに分類できる。内臓脂肪型肥満は、大網、腸間膜周囲に存在する腹腔内脂肪の蓄積量が増加する肥満で、糖尿病（特に、インスリン抵抗性を伴うＩＩ型糖尿病）、動脈硬化症、肝臓病、心臓病等を引き起こす元凶の一つとされ、現代社会において大きな問題となっている。 Obesity is a systemic increase in adipose tissue and occurs when the amount of energy consumed over a long period is greater than the amount of energy consumed. Obesity can be classified into visceral fat type obesity and subcutaneous fat type obesity. Visceral fat-type obesity is obesity in which the accumulation of intra-abdominal fat existing around the omentum and mesentery increases, and diabetes (particularly type II diabetes with insulin resistance), arteriosclerosis, liver disease, heart This is one of the main causes of illness and is a major problem in modern society.
　糖尿病は、持続的高血糖状態を伴う疾患であり、多くの環境因子と遺伝的因子とが作用した結果に生じるとされている。体内における血糖の主要な調整因子はインスリンであり、高血糖は、インスリン欠乏、又は、その作用を阻害する諸因子（例えば、遺伝的素因、運動不足、肥満、ストレス等）が過剰となって生じる。糖尿病には主として２つの種類があり、自己免疫疾患等による膵インスリン分泌機能の低下によって生じるＩ型糖尿病と、持続的な高インスリン分泌に伴う膵疲弊による膵インスリン分泌機能の低下が原因であるＩＩ型糖尿病とに分類される。日本人の糖尿病患者の９５％以上はＩＩ型糖尿病と言われており、今日、生活様式の変化に伴う患者数の増加が問題となっている。 Diabetes mellitus is a disease accompanied by a persistent hyperglycemic state, and is considered to result from the action of many environmental factors and genetic factors. The main regulator of blood glucose in the body is insulin, and hyperglycemia is caused by insulin deficiency or excessive factors that inhibit its action (eg, genetic predisposition, lack of exercise, obesity, stress, etc.) . There are mainly two types of diabetes, which are caused by type I diabetes caused by a decrease in pancreatic insulin secretion function due to autoimmune diseases and the like and a decrease in pancreatic insulin secretion function due to pancreatic exhaustion associated with continuous high insulin secretion II Classified as type 2 diabetes. More than 95% of Japanese diabetic patients are said to have type II diabetes, and today, the increase in the number of patients accompanying lifestyle changes is a problem.
　アシル－ＣｏＡ合成酵素ファミリーに属する酵素（以下、ＡＣＳと略記する）は、長鎖脂肪酸からアシルＣｏＡへ変換する酵素である。アシルＣｏＡは、細胞内脂質合成及び脂肪酸分解又は伸長反応における基質となることから、ＡＣＳは、細胞内の脂質代謝さらには脂質による細胞内シグナル伝達において中心的な役割を担う。またＡＣＳは、脂肪外脂肪酸の取込みにも関与する（非特許文献１参照）。 Enzymes belonging to the acyl-CoA synthase family (hereinafter abbreviated as ACS) are enzymes that convert long-chain fatty acids into acyl CoA. Since acyl-CoA is a substrate in intracellular lipid synthesis and fatty acid degradation or elongation reactions, ACS plays a central role in intracellular lipid metabolism as well as intracellular signaling by lipids. ACS is also involved in the uptake of extra fatty acids (see Non-Patent Document 1).
　ＡＣＳは、現在までに基質選択性や細胞内局在の異なる５つのアイソザイム（ＡＣＳ１、３、４、５、６）が同定されている（非特許文献２参照）。このファミリーに属する酵素の一つであるＡＣＳＬ１（ＧｅｎＢａｎｋ：ＮＭ_００１９９５）は、主に肝臓や脂肪組織で発現しており、トリグリセリド（ＴＧ）の合成を触媒する。なお、ＡＣＳＬ４及びＡＣＳＬ５は主に肝臓で発現し、ＡＣＳＬ３やＡＣＳＬ６は主に脳で発現していることが知られている。 As for ACS, five isozymes (ACS1, 3, 4, 5, 6) having different substrate selectivity and subcellular localization have been identified so far (see Non-Patent Document 2). ACSL1 (GenBank: NM_001995), one of the enzymes belonging to this family, is mainly expressed in the liver and adipose tissue and catalyzes the synthesis of triglyceride (TG). It is known that ACSL4 and ACSL5 are mainly expressed in the liver, and ACSL3 and ACSL6 are mainly expressed in the brain.
　ＡＣＳの阻害剤としてＴｒｉａｃｓｉｎＣが知られており、この化合物は５つのアイソザイムのうち１、３、４を阻害することが報告されている（非特許文献３参照）。この化合物については、この他にヒト肝癌細胞株であるＨｕＨ７細胞においてＴＧ蓄積を阻害すること（非特許文献４参照）やヒト正常皮膚繊維芽細胞であるＣＣＤ細胞でジアシルグリセロール、コレステロールエステル、リン脂質合成を阻害することが報告されている（非特許文献５参照）。 Triacsin C is known as an inhibitor of ACS, and this compound has been reported to inhibit 1, 3 and 4 of 5 isozymes (see Non-Patent Document 3). In addition to this compound, in addition to inhibiting TG accumulation in HuH7 cells, which are human hepatoma cell lines (see Non-Patent Document 4), and diacylglycerol, cholesterol esters, phospholipids in CCD cells, which are human normal dermal fibroblasts. It has been reported to inhibit synthesis (see Non-Patent Document 5).
　ＡＣＳＬ１は、各種癌との関係が報告されており（例えば、特許文献１～３参照）他に肝硬変や肝繊維性障害（特許文献４参照）、気管支喘息（特許文献５参照）のバイオマーカーになることも報告されている。 ACSL1 has been reported to be associated with various cancers (see, for example, Patent Documents 1 to 3), and as a biomarker for cirrhosis, liver fibrosis (see Patent Document 4), and bronchial asthma (see Patent Document 5). It has also been reported.
　また、特許文献６には、肝臓のＡＣＳＬ１の発現をｓｉＲＮＡにより抑制することによって体重の増加の抑制及び血糖値を降下させることが開示されており、ＡＣＳＬ１発現抑制剤は肥満症又はＩＩ型糖尿病の治療又は予防に利用することができることが示唆されている。 In addition, Patent Document 6 discloses that suppression of hepatic ACSL1 expression by siRNA suppresses increase in body weight and lowers blood glucose level, and ACSL1 expression inhibitor is used for obesity or type II diabetes. It has been suggested that it can be used for treatment or prevention.
　特許文献７には、ＡＣＳＬ１をターゲットとしたアンチセンス化合物について開示されている。明細書中には、３６１５のアンチセンスオリゴヌクレオチドが記載されているが、標的領域との親和性に関する予測値が記載されているだけであり、ＡＣＳＬ１発現抑制に関するデータは記載されていない。 Patent Document 7 discloses an antisense compound targeting ACSL1. In the specification, 3615 antisense oligonucleotides are described, but only predictive values regarding affinity with a target region are described, and data regarding suppression of ACSL1 expression is not described.
国際公開第０７／０１０６２８号International Publication No. 07/010628 国際公開第０７／１１７０３８号International Publication No. 07/117038 国際公開第０３／３００４９８９号International Publication No. 03/3004989 特開２００７－２５２３６６号JP 2007-252366 A 特開２００４－１２１２１８号JP 2004-121218 A 国際公開第２０１０／０７９８１９号International Publication No. 2010/0779819 国際公開第０４／０１６７４９号International Publication No. 04/016749
　本発明の目的は、優れたＡＣＳＬ１発現抑制活性を有する新規アンチセンスオリゴヌクレオチドを提供することにある。 An object of the present invention is to provide a novel antisense oligonucleotide having excellent ACSL1 expression suppression activity.
　本発明者らは、鋭意研究の結果、優れたＡＣＳＬ１発現抑制活性（ノックダウン活性）を有する新規アンチセンスオリゴヌクレオチドの合成に成功した。さらに、本発明者らは、ＡＣＳＬ１のｍＲＮＡの中で特に、アンチセンスオリゴヌクレオチドのノックダウン活性に関連する標的領域を見出した。特許文献７には、３６１５のＡＣＳＬ１に対するアンチセンスオリゴヌクレオチドが記載されているが、標的領域との親和性に関する予測値が記載されているだけであり、ＡＣＳＬ１発現抑制に関するデータは記載されていない。高親和性を持つアンチセンスが、必ずしも高い標的遺伝子発現抑制作用を持つわけではないことは技術常識である（Ａｎｔｉｓｅｎｓｅ Ｄｒｕｇ Ｔｅｃｈｎｏｌｏｇｙ Ｐｒｉｎｃｉｐｌｅｓ， Ｓｔｒａｔｅｇｉｅｓ， ａｎｄ Ａｐｐｌｉｃａｔｉｏｎｓ， ＣＲＣ Ｐｒｅｓｓ； ２ｎｄ ｅｄｉｔｉｏｎ， ２００７， ｐａｇｅ １２０－１２２， Ｆｉｇｕｒｅ ５．３ａ）。よって、特許文献７の記載からは、ＡＣＳＬ１のｍＲＮＡのどの領域がアンチセンスオリゴヌクレオチドの標的領域として有用であるかは予測できない。本発明のアンチセンスオリゴヌクレオチドは、本発明者らが見出した特定の標的領域と結合し、優れたＡＣＳＬ１発現抑制活性を示す。
　また、本発明者らは、本発明のアンチセンスオリゴヌクレオチドが、他のアイソザイム（ＡＣＳＬ３、ＡＣＳＬ４及びＡＣＳＬ５）に対して発現抑制作用を持たず、ＡＣＳＬ１特異的発現抑制作用を持つことを見出した。このように標的配列に対して特異性の高いアンチセンスオリゴヌクレオチドは、医薬として有用である。
　さらに、本発明者らは、本発明のアンチセンスオリゴヌクレオチド投与時のアスパラギン酸アミノトランスフェラーゼ（ＡＳＴ）及びアラニンアミノトランスフェラーゼ（ＡＬＴ）を測定し、肝臓毒性が見られないことを確認している。そのため、本発明のアンチセンスオリゴヌクレオチドは毒性が低く、医薬として使用するために十分安全である。
　また、本発明のアンチセンスオリゴヌクレオチドは代謝安定性及び水溶性がよく、医薬として使用するために十分安全である。 As a result of intensive studies, the present inventors have succeeded in synthesizing a novel antisense oligonucleotide having excellent ACSL1 expression suppression activity (knockdown activity). Furthermore, the present inventors have found a target region related to the knockdown activity of an antisense oligonucleotide, particularly in the mRNA of ACSL1. Patent Document 7 describes an antisense oligonucleotide for ACSL1 of 3,615, but only describes a predicted value for affinity with a target region, and does not describe data regarding suppression of ACSL1 expression. It is common knowledge that antisense having high affinity does not necessarily have a high target gene expression-inhibiting action (Antisense Drug Technology Principles, Strategies, and Applications, CRC Press; 2nd edition, 2007-120, p. , FIG. 5.3a). Therefore, from the description in Patent Document 7, it cannot be predicted which region of ACSL1 mRNA is useful as the target region of the antisense oligonucleotide. The antisense oligonucleotide of the present invention binds to a specific target region found by the present inventors and exhibits excellent ACSL1 expression suppression activity.
In addition, the present inventors have found that the antisense oligonucleotide of the present invention does not have an expression suppressing action on other isozymes (ACSL3, ACSL4 and ACSL5) and has an ACSL1-specific expression suppressing action. Thus, an antisense oligonucleotide with high specificity for a target sequence is useful as a medicine.
Furthermore, the present inventors measured aspartate aminotransferase (AST) and alanine aminotransferase (ALT) upon administration of the antisense oligonucleotide of the present invention, and confirmed that no liver toxicity was observed. Therefore, the antisense oligonucleotide of the present invention has low toxicity and is sufficiently safe for use as a medicine.
Moreover, the antisense oligonucleotide of the present invention has good metabolic stability and water solubility, and is sufficiently safe for use as a medicine.
　すなわち、本発明は、以下に関する。
（１）ストリンジェントな条件で、配列番号１の８３２位～８５３位、３６３５位～３６５４位又は３６５９位～３６７６位からなる配列にハイブリダイズ可能な配列を含む、アンチセンスオリゴヌクレオチド。
（２）ＡＣＳＬ１の発現を抑制する、（１）記載のアンチセンスオリゴヌクレオチド。
（３）長さが１３～１９塩基である、（１）又は（２）記載のアンチセンスオリゴヌクレオチド。
（４）４’位と２’位との間に架橋構造を有する糖修飾ヌクレオシドを１以上含有する、（１）～（３）いずれかに記載のアンチセンスオリゴヌクレオチド。
（５）該架橋構造が、４’－ＣＨ２－Ｏ－２’又は４’－ＣＯ－ＮＲ１－２’（Ｒ１は、水素原子又はアルキルである）である、（４）記載のアンチセンスオリゴヌクレオチド。
（６）ヌクレオシド間の１若しくは複数の結合がホスホロチオエート結合である、（１）～（５）いずれかに記載のアンチセンスオリゴヌクレオチド。
（７）配列番号３１０～３２０のいずれかに記載の配列、又は、
配列番号３１０～３２０のいずれかに記載の配列において、１若しくは数個の塩基が欠失、置換若しくは挿入された配列
を含む、（１）～（６）いずれかに記載のアンチセンスオリゴヌクレオチド。
（８）配列番号３１０～３２０のいずれかに記載の配列、又は、
配列番号３１０～３２０のいずれかに記載の配列において、１若しくは数個の塩基が欠失、置換若しくは挿入された配列
からなる、（７）記載のアンチセンスオリゴヌクレオチド。
（９）（１）～（８）いずれかに記載のアンチセンスオリゴヌクレオチドを含有する医薬組成物。
（１０）ＡＣＳＬ１が関連する疾患の予防又は治療のために用いる、（９）記載の医薬組成物。
（１１）該疾患が、肥満症又はＩＩ型糖尿病である、（１０）記載の医薬組成物。 That is, the present invention relates to the following.
(1) An antisense oligonucleotide comprising a sequence capable of hybridizing to a sequence consisting of positions 832 to 853, 3635 to 3654, or 3659 to 3676 of SEQ ID NO: 1 under stringent conditions.
(2) The antisense oligonucleotide according to (1), which suppresses the expression of ACSL1.
(3) The antisense oligonucleotide according to (1) or (2), which is 13 to 19 bases in length.
(4) The antisense oligonucleotide according to any one of (1) to (3), which contains at least one sugar-modified nucleoside having a cross-linked structure between the 4′-position and the 2′-position.
(5) The cross-linking structure is 4′-CH 2 —O-2 ′ or 4′-CO—NR 1 -2 ′ (R 1 is a hydrogen atom or alkyl), Sense oligonucleotide.
(6) The antisense oligonucleotide according to any one of (1) to (5), wherein one or more bonds between nucleosides are phosphorothioate bonds.
(7) the sequence described in any of SEQ ID NOS: 310 to 320, or
The antisense oligonucleotide according to any one of (1) to (6), comprising a sequence in which one or several bases are deleted, substituted or inserted in the sequence described in any of SEQ ID NOS: 310 to 320.
(8) the sequence described in any of SEQ ID NOS: 310 to 320, or
The antisense oligonucleotide according to (7), which consists of a sequence described in any one of SEQ ID NOS: 310 to 320, wherein one or several bases are deleted, substituted, or inserted.
(9) A pharmaceutical composition comprising the antisense oligonucleotide according to any one of (1) to (8).
(10) The pharmaceutical composition according to (9), which is used for prevention or treatment of a disease associated with ACSL1.
(11) The pharmaceutical composition according to (10), wherein the disease is obesity or type II diabetes.
　さらに、　以下の発明も本発明に含まれる。
（１－１）配列番号３２１～３６２のいずれかに記載の配列、又は、
配列番号３２１～３６２のいずれかに記載の配列において、１若しくは数個の塩基が欠失、置換若しくは挿入された配列
を含む、アンチセンスオリゴヌクレオチド。
（１－２）ＡＣＳＬ１の発現を抑制する、（１－１）記載のアンチセンスオリゴヌクレオチド。
（１－３）長さが１３～１９塩基である、（１－１）又は（１－２）記載のアンチセンスオリゴヌクレオチド。
（１－４）配列番号３２１～３６２のいずれかに記載の配列、又は、
配列番号３２１～３６２のいずれかに記載の配列において、１若しくは数個の塩基が欠失、置換若しくは挿入された配列
からなる、（１－１）～（１－３）いずれかに記載のアンチセンスオリゴヌクレオチド。
（１－５）４’位と２’位との間に架橋構造を有する糖修飾ヌクレオシドを１以上含有する、（１－１）～（１－４）いずれかに記載のアンチセンスオリゴヌクレオチド。
（１－６）該架橋構造が、４’－ＣＨ２－Ｏ－２’又は４’－ＣＯ－ＮＲ１－２’（Ｒ１は、水素原子又はアルキルである）である、（１－５）記載のアンチセンスオリゴヌクレオチド。
（１－７）ヌクレオシド間の１若しくは複数の結合がホスホロチオエート結合である、（１－１）～（１－６）いずれかに記載のアンチセンスオリゴヌクレオチド。
（１－８）（１－１）～（１－７）いずれかに記載のアンチセンスオリゴヌクレオチドを含有する医薬組成物。
（１－９）ＡＣＳＬ１が関連する疾患の予防又は治療のために用いる、（１－８）記載の医薬組成物。
（１－１０）該疾患が、肥満症又はＩＩ型糖尿病である、（１－９）記載の医薬組成物。 Furthermore, the following invention is also included in the present invention.
(1-1) the sequence described in any one of SEQ ID NOs: 321 to 362, or
An antisense oligonucleotide comprising a sequence according to any one of SEQ ID NOs: 321 to 362, wherein one or several bases are deleted, substituted or inserted.
(1-2) The antisense oligonucleotide according to (1-1), which suppresses the expression of ACSL1.
(1-3) The antisense oligonucleotide according to (1-1) or (1-2), which has a length of 13 to 19 bases.
(1-4) the sequence described in any one of SEQ ID NOS: 321 to 362, or
The anti-sequence according to any one of (1-1) to (1-3), comprising a sequence in which one or several bases are deleted, substituted or inserted in the sequence described in any of SEQ ID NOs: 321 to 362. Sense oligonucleotide.
(1-5) The antisense oligonucleotide according to any one of (1-1) to (1-4), which contains one or more sugar-modified nucleosides having a cross-linked structure between the 4 ′ position and the 2 ′ position.
(1-6) The crosslinked structure is 4′-CH 2 —O-2 ′ or 4′-CO—NR 1 -2 ′ (R 1 is a hydrogen atom or alkyl) (1-5 ) The antisense oligonucleotide described.
(1-7) The antisense oligonucleotide according to any one of (1-1) to (1-6), wherein one or more bonds between nucleosides are phosphorothioate bonds.
(1-8) A pharmaceutical composition comprising the antisense oligonucleotide according to any one of (1-1) to (1-7).
(1-9) The pharmaceutical composition according to (1-8), which is used for prevention or treatment of a disease associated with ACSL1.
(1-10) The pharmaceutical composition according to (1-9), wherein the disease is obesity or type II diabetes.
　さらに、　以下の発明も本発明に含まれる。
（２－１）配列番号９０、１３３、１６４、１９４、２２１、２３４、２４４、２５４、２６７、２７３、２８０、２８４、２８５、２８６、２９３若しくは３０２のいずれかに記載の配列、又は、
配列番号９０、１３３、１６４、１９４、２２１、２３４、２４４、２５４、２６７、２７３、２８０、２８４、２８５、２８６、２９３若しくは３０２のいずれかに記載の配列において、１若しくは数個の塩基が欠失、置換若しくは挿入された配列
を含む、アンチセンスオリゴヌクレオチド。
（２－２）Ａｃｓｌ１の発現を抑制する、（２－１）記載のアンチセンスオリゴヌクレオチド。
（２－３）長さが１３～１９塩基である、（２－１）又は（２－２）記載のアンチセンスオリゴヌクレオチド。
（２－４）配列番号９０、１３３、１６４、１９４、２２１、２３４、２４４、２５４、２６７、２７３、２８０、２８４、２８５、２８６、２９３若しくは３０２のいずれかに記載の配列、又は、
配列番号９０、１３３、１６４、１９４、２２１、２３４、２４４、２５４、２６７、２７３、２８０、２８４、２８５、２８６、２９３若しくは３０２のいずれかに記載の配列において、１若しくは数個の塩基が欠失、置換若しくは挿入された配列
からなる、（２－１）～（２－３）いずれかに記載のアンチセンスオリゴヌクレオチド。
（２－５）４’位と２’位との間に架橋構造を有する糖修飾ヌクレオシドを１以上含有する、（２－１）～（２－４）いずれかに記載のアンチセンスオリゴヌクレオチド。
（２－６）該架橋構造が、４’－ＣＨ２－Ｏ－２’又は４’－ＣＯ－ＮＲ１－２’（Ｒ１は、水素原子又はアルキルである）である、（２－５）記載のアンチセンスオリゴヌクレオチド。
（２－７）ヌクレオシド間の１若しくは複数の結合がホスホロチオエート結合である、（２－１）～（２－６）いずれかに記載のアンチセンスオリゴヌクレオチド。
（２－８）（２－１）～（２－７）いずれかに記載のアンチセンスオリゴヌクレオチドを含有する医薬組成物。
（２－９）ＡＣＳＬ１が関連する疾患の予防又は治療のために用いる、（２－８）記載の医薬組成物。
（２－１０）該疾患が、肥満症又はＩＩ型糖尿病である、（２－９）記載の医薬組成物。 Furthermore, the following invention is also included in the present invention.
(2-1) SEQ ID NO: 90, 133, 164, 194, 221, 234, 244, 254, 267, 273, 280, 284, 285, 286, 293 or 302, or
SEQ ID NO: 90, 133, 164, 194, 221, 234, 244, 254, 267, 273, 280, 284, 285, 286, 293 or 302 lacks one or several bases. An antisense oligonucleotide comprising a missing, substituted or inserted sequence.
(2-2) The antisense oligonucleotide according to (2-1), which suppresses the expression of Acsl1.
(2-3) The antisense oligonucleotide according to (2-1) or (2-2), which is 13 to 19 bases in length.
(2-4) the sequence according to any of SEQ ID NOs: 90, 133, 164, 194, 221, 234, 244, 254, 267, 273, 280, 284, 285, 286, 293 or 302, or
SEQ ID NO: 90, 133, 164, 194, 221, 234, 244, 254, 267, 273, 280, 284, 285, 286, 293 or 302 lacks one or several bases. The antisense oligonucleotide according to any one of (2-1) to (2-3), which comprises a deleted, substituted or inserted sequence.
(2-5) The antisense oligonucleotide according to any one of (2-1) to (2-4), which contains one or more sugar-modified nucleosides having a cross-linked structure between the 4 ′ position and the 2 ′ position.
(2-6) The crosslinked structure is 4′-CH 2 —O-2 ′ or 4′-CO—NR 1 -2 ′ (R 1 is a hydrogen atom or alkyl) (2-5 ) The antisense oligonucleotide described.
(2-7) The antisense oligonucleotide according to any one of (2-1) to (2-6), wherein one or more bonds between nucleosides are phosphorothioate bonds.
(2-8) A pharmaceutical composition comprising the antisense oligonucleotide according to any one of (2-1) to (2-7).
(2-9) The pharmaceutical composition according to (2-8), which is used for prevention or treatment of a disease associated with ACSL1.
(2-10) The pharmaceutical composition according to (2-9), wherein the disease is obesity or type II diabetes.
　さらに、　以下の発明も本発明に含まれる。
（３－１）配列番号３６３、３６５～３６９、３７５～３７８、３８０～３８９、３９２～３９４、３９８、４００～４０３、４０５、４０８～４１３、４１５～４１８、４２３～４２５、４２８、４３０～４３３、４３６、４３７、４４０、４４３～４４７、４５０、４５２、４５３、４５６～４５８、４６２、４６４～４６９、４７３～４７５、４７７、４７８、４８０～４８２、４８４～４８６、４８９、４９１～４９８、５００若しくは５０２のいずれかに記載の配列、又は、
配列番号３６３、３６５～３６９、３７５～３７８、３８０～３８９、３９２～３９４、３９８、４００～４０３、４０５、４０８～４１３、４１５～４１８、４２３～４２５、４２８、４３０～４３３、４３６、４３７、４４０、４４３～４４７、４５０、４５２、４５３、４５６～４５８、４６２、４６４～４６９、４７３～４７５、４７７、４７８、４８０～４８２、４８４～４８６、４８９、４９１～４９８、５００若しくは５０２のいずれかに記載の配列において、１若しくは数個の塩基が欠失、置換若しくは挿入された配列
を含む、アンチセンスオリゴヌクレオチド。
（３－２）ＡＣＳＬ１の発現を抑制する、（３－１）記載のアンチセンスオリゴヌクレオチド。
（３－３）長さが１３～１９塩基である、（３－１）又は（３－２）記載のアンチセンスオリゴヌクレオチド。
（３－４）配列番号３６３、３６５～３６９、３７５～３７８、３８０～３８９、３９２～３９４、３９８、４００～４０３、４０５、４０８～４１３、４１５～４１８、４２３～４２５、４２８、４３０～４３３、４３６、４３７、４４０、４４３～４４７、４５０、４５２、４５３、４５６～４５８、４６２、４６４～４６９、４７３～４７５、４７７、４７８、４８０～４８２、４８４～４８６、４８９、４９１～４９８、５００若しくは５０２のいずれかに記載の配列、又は、
配列番号３６３、３６５～３６９、３７５～３７８、３８０～３８９、３９２～３９４、３９８、４００～４０３、４０５、４０８～４１３、４１５～４１８、４２３～４２５、４２８、４３０～４３３、４３６、４３７、４４０、４４３～４４７、４５０、４５２、４５３、４５６～４５８、４６２、４６４～４６９、４７３～４７５、４７７、４７８、４８０～４８２、４８４～４８６、４８９、４９１～４９８、５００若しくは５０２のいずれかに記載の配列において、１若しくは数個の塩基が欠失、置換若しくは挿入された配列
からなる、（３－１）～（３－３）いずれかに記載のアンチセンスオリゴヌクレオチド。
（３－５）４’位と２’位との間に架橋構造を有する糖修飾ヌクレオシドを１以上含有する、（３－１）～（３－４）いずれかに記載のアンチセンスオリゴヌクレオチド。
（３－６）該架橋構造が、４’－ＣＨ２－Ｏ－２’又は４’－ＣＯ－ＮＲ１－２’（Ｒ１は、水素原子又はアルキルである）である、（１－５）記載のアンチセンスオリゴヌクレオチド。
（３－７）ヌクレオシド間の１若しくは複数の結合がホスホロチオエート結合である、（３－１）～（３－６）いずれかに記載のアンチセンスオリゴヌクレオチド。
（３－８）（３－１）～（３－７）いずれかに記載のアンチセンスオリゴヌクレオチドを含有する医薬組成物。
（３－９）ＡＣＳＬ１が関連する疾患の予防又は治療のために用いる、（３－８）記載の医薬組成物。
（３－１０）該疾患が、肥満症又はＩＩ型糖尿病である、（３－９）記載の医薬組成物。 Furthermore, the following invention is also included in the present invention.
(3-1) SEQ ID NOS: 363, 365-369, 375-378, 380-389, 392-394, 398, 400-403, 405, 408-413, 415-418, 423-425, 428, 430-433 436, 437, 440, 443-447, 450, 452, 453, 456-458, 462, 464-469, 473-475, 477, 478, 480-482, 484-486, 489, 491-498, 500 Or the sequence according to any one of 502, or
SEQ ID NOs: 363, 365-369, 375-378, 380-389, 392-394, 398, 400-403, 405, 408-413, 415-418, 423-425, 428, 430-433, 436, 437, 440, 443-447, 450, 452, 453, 456-458, 462, 464-469, 473-475, 477, 478, 480-482, 484-486, 489, 491-498, 500 or 502 An antisense oligonucleotide comprising a sequence wherein one or several bases are deleted, substituted or inserted in the sequence described in 1.
(3-2) The antisense oligonucleotide according to (3-1), which suppresses the expression of ACSL1.
(3-3) The antisense oligonucleotide according to (3-1) or (3-2), which has a length of 13 to 19 bases.
(3-4) SEQ ID NOS: 363, 365-369, 375-378, 380-389, 392-394, 398, 400-403, 405, 408-413, 415-418, 423-425, 428, 430-433 436, 437, 440, 443-447, 450, 452, 453, 456-458, 462, 464-469, 473-475, 477, 478, 480-482, 484-486, 489, 491-498, 500 Or the sequence according to any one of 502, or
SEQ ID NOs: 363, 365-369, 375-378, 380-389, 392-394, 398, 400-403, 405, 408-413, 415-418, 423-425, 428, 430-433, 436, 437, 440, 443-447, 450, 452, 453, 456-458, 462, 464-469, 473-475, 477, 478, 480-482, 484-486, 489, 491-498, 500 or 502 The antisense oligonucleotide according to any one of (3-1) to (3-3), comprising a sequence in which one or several bases are deleted, substituted or inserted in the sequence described in (1).
(3-5) The antisense oligonucleotide according to any one of (3-1) to (3-4), which contains one or more sugar-modified nucleosides having a cross-linked structure between the 4 ′ position and the 2 ′ position.
(3-6) The crosslinked structure is 4′-CH 2 —O-2 ′ or 4′-CO—NR 1 -2 ′ (R 1 is a hydrogen atom or alkyl) (1-5 ) The antisense oligonucleotide described.
(3-7) The antisense oligonucleotide according to any one of (3-1) to (3-6), wherein one or more bonds between nucleosides are phosphorothioate bonds.
(3-8) A pharmaceutical composition comprising the antisense oligonucleotide according to any one of (3-1) to (3-7).
(3-9) The pharmaceutical composition according to (3-8), which is used for prevention or treatment of a disease associated with ACSL1.
(3-10) The pharmaceutical composition according to (3-9), wherein the disease is obesity or type II diabetes.
　本発明のアンチセンスオリゴヌクレオチドは優れたＡＣＳＬ１発現抑制活性を示し、医薬品、特にＡＣＳＬ１の関与する疾患、例えば、肥満症、肥満関連疾患、糖尿病（特に、ＩＩ型糖尿病）、シンドロームＸ、心臓血管障害又は癌（乳癌、結腸癌、大腸癌、卵巣癌、肺癌等）の予防又は治療のための医薬（体重管理用医薬も含む）として非常に有用である。 The antisense oligonucleotide of the present invention exhibits excellent ACSL1 expression-suppressing activity, and is associated with drugs, particularly diseases involving ACSL1, such as obesity, obesity-related diseases, diabetes (particularly type II diabetes), syndrome X, cardiovascular disorders Or it is very useful as a medicine (including a medicine for weight management) for preventing or treating cancer (breast cancer, colon cancer, colon cancer, ovarian cancer, lung cancer, etc.).
本発明のアンチセンスオリゴヌクレオチドを投与した際の肝臓中Ａｃｓｌ１タンパク質量解析　（初回投与後２８日）Analysis of the amount of Acsl1 protein in the liver when the antisense oligonucleotide of the present invention was administered (28 days after the first administration) 本発明のアンチセンスオリゴヌクレオチドを投与した際の体重推移Change in body weight when the antisense oligonucleotide of the present invention is administered 本発明のアンチセンスオリゴヌクレオチドを投与した際の摂餌量変化Change in food intake upon administration of the antisense oligonucleotide of the present invention
　本明細書において使用される用語は、特に言及する場合を除いて、当該分野で通常用いられる意味で用いられる。
　本発明においては、当該分野で公知の遺伝子操作方法の使用が可能である。例えば、Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ，　Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，　Ｔｈｉｒｄ　Ｅｄｉｔｉｏｎ，　Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ　（２００１）、Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，　Ｊｏｈｎ　Ｗｉｌｅｙ　＆　Ｓｏｎｓ　（２００３）に記載された方法等が挙げられる。
　以下に本発明について詳細に説明する。 The terms used in the present specification are used in the meaning normally used in the art unless otherwise specified.
In the present invention, a genetic manipulation method known in the art can be used. For example, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001), Current Protocols in Molecular Biology, Jon.
The present invention is described in detail below.
　「アンチセンスオリゴヌクレオチド」（ＡＯＮ）とは、標的遺伝子のｍＲＮＡ、ｍＲＮＡ前駆体又はｎｃＲＮＡに対して相補的なオリゴヌクレオチドであり、１本鎖のＤＮＡ、ＲＮＡ及び／又はそれらの類似体から構成される。当該アンチセンスオリゴヌクレオチドが標的とするｍＲＮＡ、ｍＲＮＡ前駆体又はｎｃＲＮＡと二本鎖を形成することによりｍＲＮＡ、ｍＲＮＡ前駆体又はｎｃＲＮＡの働きを抑制する。「アンチセンスオリゴヌクレオチド」には、標的となるｍＲＮＡ、ｍＲＮＡ前駆体又はｎｃＲＮＡと完全に相補的であるもののみならず、ｍＲＮＡ、ｍＲＮＡ前駆体又はｎｃＲＮＡとストリンジェントな条件でハイブリダイズできる限り、１若しくは数個のミスマッチが存在するものも含まれる。
　オリゴヌクレオチドとは、同一又は異なるヌクレオシドが複数個結合したヌクレオチドを意味する。「ヌクレオシド」とは、核酸塩基と糖とがＮ－グリコシド結合をした化合物を意味する。「ヌクレオチド」とは、ヌクレオシドの糖にリン酸基が結合した化合物を意味する。
　ＤＮＡ又はＲＮＡの類似体とは、ＤＮＡ又はＲＮＡに似た構造を持つ分子を意味する。例えば、ペプチド核酸（ＰＮＡ）等が挙げられる。
　ｎｃＲＮＡ（ノンコーディングＲＮＡ）とは、タンパク質へ翻訳されずに機能するＲＮＡの総称である。例えば、リボソームＲＮＡ、転移ＲＮＡ、ｍｉＲＮＡ等が挙げられる。
　１若しくは数個のミスマッチとは、１～５個、好ましくは１～３個、さらに好ましくは１又は２個のミスマッチを意味している。 An “antisense oligonucleotide” (AON) is an oligonucleotide complementary to a target gene mRNA, mRNA precursor or ncRNA, and is composed of single-stranded DNA, RNA and / or analogs thereof. The The function of mRNA, mRNA precursor or ncRNA is suppressed by forming a double strand with the mRNA, mRNA precursor or ncRNA targeted by the antisense oligonucleotide. “Antisense oligonucleotides” include not only those that are completely complementary to the target mRNA, mRNA precursor or ncRNA, but also those that can hybridize to the mRNA, mRNA precursor or ncRNA under stringent conditions. Or, there are cases where several mismatches exist.
An oligonucleotide means a nucleotide in which a plurality of identical or different nucleosides are bound. “Nucleoside” means a compound in which a nucleobase and a sugar form an N-glycoside bond. “Nucleotide” means a compound in which a phosphate group is bonded to a nucleoside sugar.
An analog of DNA or RNA means a molecule having a structure similar to DNA or RNA. For example, peptide nucleic acid (PNA) etc. are mentioned.
ncRNA (non-coding RNA) is a general term for RNA that functions without being translated into protein. For example, ribosomal RNA, transfer RNA, miRNA and the like can be mentioned.
One or several mismatches means 1 to 5, preferably 1 to 3, more preferably 1 or 2 mismatches.
　本発明のアンチセンスオリゴヌクレオチドの標的遺伝子としては、ＡＣＳＬ１が挙げられる。例えば、ヒトＡＣＳＬ１、マウスＡｃｓｌ１等が挙げられるが、これらに限定されない。 ACSL1 is mentioned as a target gene of the antisense oligonucleotide of the present invention. For example, human ACSL1, mouse Acs11 and the like can be mentioned, but not limited thereto.
　「ＡＣＳＬ１」は公知のタンパク質である。ヒトＡＣＳＬ１（ＧｅｎＢａｎｋ：ＮＭ_００１９９５）のＤＮＡ配列を配列表の配列番号１に、アミノ酸配列を配列番号２に記載する。マウスＡｃｓｌ１（ＧｅｎＢａｎｋ：ＮＭ_００７９８１）のＤＮＡ配列を配列表の配列番号３に、アミノ酸配列を配列番号４に記載する。本発明における「ＡＣＳＬ１」は、これらの配列に限定されるものではなく、配列番号２又は４のタンパク質の機能が保持される限り、アミノ酸やＤＮＡの変異数や変異部位に制限はないものとする。 “ACSL1” is a known protein. The DNA sequence of human ACSL1 (GenBank: NM — 001995) is described in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence is described in SEQ ID NO: 2. The DNA sequence of mouse Acsl1 (GenBank: NM — 007981) is described in SEQ ID NO: 3 and the amino acid sequence is described in SEQ ID NO: 4. “ACSL1” in the present invention is not limited to these sequences, and as long as the function of the protein of SEQ ID NO: 2 or 4 is maintained, the number of amino acid and DNA mutations and mutation sites are not limited. .
　本発明のアンチセンスオリゴヌクレオチドの長さは、６～５０塩基である。例えば、６～３０塩基、６～１９塩基、８～１９塩基、１０～１９塩基、１３～１９塩基、１３塩基、１４塩基、１５塩基、１６塩基、１７塩基、１８塩基、１９塩基である。 The length of the antisense oligonucleotide of the present invention is 6 to 50 bases. For example, 6 to 30 bases, 6 to 19 bases, 8 to 19 bases, 10 to 19 bases, 13 to 19 bases, 13 bases, 14 bases, 15 bases, 16 bases, 17 bases, 18 bases and 19 bases.
　本発明の「アンチセンスオリゴヌクレオチド」としては、
（ａ）ストリンジェントな条件で、配列番号１の８３２位～８５３位、３６３５位～３６５４位若しくは３６５９位～３６７６位からなる配列にハイブリダイズ可能な配列を含む、アンチセンスオリゴヌクレオチド、又は
（ｂ）ストリンジェントな条件で、配列番号３の８８３位～８９９位、３７１８位～３７３７位若しくは３７４１位～３７５９位からなる配列にハイブリダイズ可能な配列を含む、アンチセンスオリゴヌクレオチド
が挙げられる。（ａ）の標的領域は、それぞれ、ヒトＡＣＳＬ１のｍＲＮＡの中で特に、アンチセンスオリゴヌクレオチドのノックダウン活性に関連する領域である。（ｂ）の標的領域は、それぞれ、マウスＡｃｓｌ１のｍＲＮＡの中で特に、アンチセンスオリゴヌクレオチドのノックダウン活性に関連する領域である。ストリンジェントな条件で、該標的領域にハイブリダイズ可能な配列であれば、長さやヌクレオチドの修飾の有無に関わらず、本発明のアンチセンスオリゴヌクレオチドに含まれる。 As the “antisense oligonucleotide” of the present invention,
(A) an antisense oligonucleotide comprising a sequence hybridizable to a sequence consisting of positions 832 to 853, 3635 to 3654, or 3659 to 3676 of SEQ ID NO: 1 under stringent conditions; or (b ) An antisense oligonucleotide containing a sequence that can hybridize to the sequence consisting of positions 883 to 899, 3718 to 3737, or 3741 to 3759 of SEQ ID NO: 3 under stringent conditions. Each of the target regions in (a) is a region associated with the knockdown activity of the antisense oligonucleotide, particularly in human ACSL1 mRNA. Each of the target regions in (b) is a region associated with the knockdown activity of the antisense oligonucleotide, particularly in the mouse Acsl1 mRNA. Any sequence capable of hybridizing to the target region under stringent conditions is included in the antisense oligonucleotide of the present invention regardless of the length or the presence or absence of nucleotide modification.
　ＡＣＳＬ１発現抑制活性（ノックダウン活性）は、公知の方法により測定することが可能である。例えば、後述する実施例４～実施例７記載の方法により測定することができる。 ACSL1 expression inhibitory activity (knockdown activity) can be measured by a known method. For example, it can be measured by the method described in Examples 4 to 7 described later.
　本発明のアンチセンスオリゴヌクレオチドとして、具体的には、
（ａ）配列番号１の８３２位～８５３位、３６３５位～３６５４位若しくは３６５９位～３６７６位からなる配列、又は
（ｂ）配列番号３の８８３位～８９９位、３７１８位～３７３７位若しくは３７４１位～３７５９位からなる配列
の相補配列と少なくとも７０％以上、好ましくは８０％以上、より好ましくは９０％以上、もっとも好ましくは９５％以上の相同性を有するアンチセンスオリゴヌクレオチドが挙げられる。ここで、相同性は、例えば、Ａｌｔｓｃｈｕｌら（Ｔｈｅ　Ｊｏｕｒｎａｌ　ｏｆ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，２１５，４０３－４１０（１９９０）．）の開発したアルゴリズムを使用した検索プログラムＢＬＡＳＴを用いることにより、スコアで類似度が示される。
　「ストリンジェントな条件」とは、アンチセンスオリゴヌクレオチドのみが、
（ａ）配列番号１の８３２位～８５３位、３６３５位～３６５４位若しくは３６５９位～３６７６位からなる配列、又は
（ｂ）配列番号３の８８３位～８９９位、３７１８位～３７３７位若しくは３７４１位～３７５９位からなる配列
とハイブリット（いわゆる特異的ハイブリット）を形成し、同等の機能を有しない塩基配列は該特定配列とハイブリット（いわゆる非特異的ハイブリット）を形成しない条件を意味する。当業者は、ハイブリダイゼーション反応及び洗浄時の温度や、ハイブリダイゼーション反応液及び洗浄液の塩濃度等を変化させることによって、このような条件を容易に選択することができる。具体的には、６×ＳＳＣ（０．９Ｍ　ＮａＣｌ，０．０９Ｍ　クエン酸三ナトリウム）又は６×ＳＳＰＥ（３Ｍ　ＮａＣｌ，０，２Ｍ　ＮａＨ２ＰＯ４，２０ｍＭ　ＥＤＴＡ・２Ｎａ，ｐＨ７．４）中４２℃でハイブリダイズさせ、さらに４２℃で０．５×ＳＳＣにより洗浄する条件が、本発明のストリンジェントな条件の１例として挙げられるが、これに限定されるものではない。ハイブリダイゼーション方法としては、当該分野において周知慣用な手法、例えば、サザンブロットハイブリダイゼーション法等を用いることができる。具体的には、Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ：Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，Ｓｅｃｏｎｄ　Ｅｄｉｔｉｏｎ（１９８９）（Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ）、Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ（１９９４）（Ｗｉｌｅｙ－Ｉｎｔｅｒｓｃｉｅｎｃｅ）、ＤＮＡ　Ｃｌｏｎｉｎｇ　１：Ｃｏｒｅ　Ｔｅｃｈｎｉｑｕｅｓ、Ａ　Ｐｒａｃｔｉｃａｌ　Ａｐｐｒｏａｃｈ，Ｓｅｃｏｎｄ　Ｅｄｉｔｉｏｎ（１９９５）（Ｏｘｆｏｒｄ　Ｕｎｉｖｅｒｓｉｔｙ　Ｐｒｅｓｓ）等に記載されている方法に準じて行うことができる。 As the antisense oligonucleotide of the present invention, specifically,
(A) a sequence consisting of positions 832 to 853, 3635 to 3654 or 3659 to 3676 of SEQ ID NO: 1, or (b) 883 to 899, 3718 to 3737 or 3741 of SEQ ID NO: 3. An antisense oligonucleotide having at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more homology with a complementary sequence of the sequence consisting of ˜3759 positions. Here, the homology is indicated in the score by using a search program BLAST using an algorithm developed by Altschul et al. (The Journal of Molecular Biology, 215, 403-410 (1990)), for example. .
“Stringent conditions” means that only antisense oligonucleotides
(A) a sequence consisting of positions 832 to 853, 3635 to 3654 or 3659 to 3676 of SEQ ID NO: 1, or (b) 883 to 899, 3718 to 3737 or 3741 of SEQ ID NO: 3. A base sequence that does not form a hybrid (so-called non-specific hybrid) with the specific sequence means that it forms a hybrid (so-called specific hybrid) with the sequence consisting of ˜3759 and does not have an equivalent function. Those skilled in the art can easily select such conditions by changing the temperature during the hybridization reaction and washing, the salt concentration of the hybridization reaction solution and the washing solution, and the like. Specifically, it is 42 ° C. in 6 × SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6 × SSPE (3 M NaCl, 0, 2 M NaH 2 PO 4 , 20 mM EDTA · 2Na, pH 7.4). Examples of the stringent conditions of the present invention include, but are not limited to, the conditions in which the cells are hybridized with and washed with 0.5 × SSC at 42 ° C. As a hybridization method, a well-known and commonly used method in this field, for example, a Southern blot hybridization method or the like can be used. Specifically, Molecular Cloning: A Laboratory Manual, Second Edition (1989) (Cold Spring Harbor Laboratory Press), Current Protocol in Amplification in Coal (1994) (Ten-Proc. It can be carried out according to the method described in Second Edition (1995) (Oxford University Press) and the like.
　本発明のアンチセンスオリゴヌクレオチドとしては、例えば、
（Ａ）配列番号３１０～３２０のいずれかに記載の配列、又は、
配列番号３１０～３２０のいずれかに記載の配列において、１若しくは数個の塩基が欠失、置換若しくは挿入された配列、
（Ｂ）配列番号３２１～３６２のいずれかに記載の配列、又は、
配列番号３２１～３６２のいずれかに記載の配列において、１若しくは数個の塩基が欠失、置換若しくは挿入された配列、
（Ｃ）配列番号３６３、３６５～３６９、３７５～３７８、３８０～３８９、３９２～３９４、３９８、４００～４０３、４０５、４０８～４１３、４１５～４１８、４２３～４２５、４２８、４３０～４３３、４３６、４３７、４４０、４４３～４４７、４５０、４５２、４５３、４５６～４５８、４６２、４６４～４６９、４７３～４７５、４７７、４７８、４８０～４８２、４８４～４８６、４８９、４９１～４９８、５００若しくは５０２のいずれかに記載の配列、又は、
配列番号３６３、３６５～３６９、３７５～３７８、３８０～３８９、３９２～３９４、３９８、４００～４０３、４０５、４０８～４１３、４１５～４１８、４２３～４２５、４２８、４３０～４３３、４３６、４３７、４４０、４４３～４４７、４５０、４５２、４５３、４５６～４５８、４６２、４６４～４６９、４７３～４７５、４７７、４７８、４８０～４８２、４８４～４８６、４８９、４９１～４９８、５００若しくは５０２のいずれかに記載の配列において、１若しくは数個の塩基が欠失、置換若しくは挿入された配列
のいずれかを含むアンチセンスオリゴヌクレオチドが挙げられる。さらに好ましくは、
（Ａ）配列番号３１０～３２０のいずれかに記載の配列、又は、
配列番号３１０～３２０のいずれかに記載の配列において、１若しくは数個の塩基が欠失、置換若しくは挿入された配列、
（Ｂ‐１）配列番号３５６又は３５８に記載の配列、又は、
配列番号３５６又は３５８に記載の配列において、１若しくは数個の塩基が欠失、置換若しくは挿入された配列、
（Ｃ－１）配列番号３８３、４００、４１７、４３２、４３３、４４４、４４６、４４７、４５８、４６４、４６５、４８２、４８６、４９２若しくは４９７に記載の配列、又は、
配列番号３８３、４００、４１７、４３２、４３３、４４４、４４６、４４７、４５８、４６４、４６５、４８２、４８６、４９２若しくは４９７に記載の配列において、１若しくは数個の塩基が欠失、置換若しくは挿入された配列を含むアンチセンスオリゴヌクレオチドが挙げられる。
これらはそれぞれ、ヒトＡＣＳＬ１発現抑制活性を有する。該配列を含む限り、長さやヌクレオチドの修飾の有無に関わらず、本発明のアンチセンスオリゴヌクレオチドに含まれる。
　さらに、本発明のアンチセンスオリゴヌクレオチドとしては、例えば、
配列番号９０、１３３、１６４、１９４、２２１、２３４、２４４、２５４、２６７、２７３、２８０、２８４、２８５、２８６、２９３若しくは３０２のいずれかに記載の配列、又は、
配列番号９０、１３３、１６４、１９４、２２１、２３４、２４４、２５４、２６７、２７３、２８０、２８４、２８５、２８６、２９３若しくは３０２のいずれかに記載の配列において、１若しくは数個の塩基が欠失、置換若しくは挿入された配列
を含むアンチセンスオリゴヌクレオチドが挙げられる。さらに好ましくは、
配列番号１３３、１６４、２４４、２８０、２８５若しくは２８６のいずれかに記載の配列、又は、
配列番号１３３、１６４、２４４、２８０、２８５若しくは２８６のいずれかに記載の配列において、１若しくは数個の塩基が欠失、置換若しくは挿入された配列
を含むアンチセンスオリゴヌクレオチドが挙げられる。これらはそれぞれ、マウスＡｃｓｌ１発現抑制活性を有する。該配列を含む限り、長さやヌクレオチドの修飾の有無に関わらず、本発明のアンチセンスオリゴヌクレオチドに含まれる。 As the antisense oligonucleotide of the present invention, for example,
(A) the sequence described in any of SEQ ID NOS: 310 to 320, or
A sequence in which one or several bases are deleted, substituted or inserted in the sequence shown in any of SEQ ID NOS: 310 to 320;
(B) the sequence described in any one of SEQ ID NOs: 321 to 362, or
A sequence in which one or several bases are deleted, substituted or inserted in the sequence of any one of SEQ ID NOs: 321 to 362,
(C) SEQ ID NOs: 363, 365-369, 375-378, 380-389, 392-394, 398, 400-403, 405, 408-413, 415-418, 423-425, 428, 430-433, 436 437, 440, 443-447, 450, 452, 453, 456-458, 462, 464-469, 473-475, 477, 478, 480-482, 484-486, 489, 491-498, 500 or 502 The sequence according to any one of
SEQ ID NOs: 363, 365-369, 375-378, 380-389, 392-394, 398, 400-403, 405, 408-413, 415-418, 423-425, 428, 430-433, 436, 437, 440, 443-447, 450, 452, 453, 456-458, 462, 464-469, 473-475, 477, 478, 480-482, 484-486, 489, 491-498, 500 or 502 In the sequence described in (1), an antisense oligonucleotide containing any one of the sequences in which one or several bases are deleted, substituted or inserted is mentioned. More preferably,
(A) the sequence described in any of SEQ ID NOS: 310 to 320, or
A sequence in which one or several bases are deleted, substituted or inserted in the sequence shown in any of SEQ ID NOS: 310 to 320;
(B-1) the sequence of SEQ ID NO: 356 or 358, or
A sequence in which one or several bases are deleted, substituted or inserted in the sequence set forth in SEQ ID NO: 356 or 358;
(C-1) the sequence of SEQ ID NO: 383, 400, 417, 432, 433, 444, 446, 447, 458, 464, 465, 482, 486, 492 or 497, or
In the sequence described in SEQ ID NOs: 383, 400, 417, 432, 433, 444, 446, 447, 458, 464, 465, 482, 486, 492, or 497, one or several bases are deleted, substituted or inserted And antisense oligonucleotides containing the sequence of interest.
Each of these has human ACSL1 expression inhibitory activity. As long as the sequence is included, it is included in the antisense oligonucleotide of the present invention regardless of the length or nucleotide modification.
Furthermore, as the antisense oligonucleotide of the present invention, for example,
The sequence according to any of SEQ ID NOs: 90, 133, 164, 194, 221, 234, 244, 254, 267, 273, 280, 284, 285, 286, 293 or 302, or
SEQ ID NO: 90, 133, 164, 194, 221, 234, 244, 254, 267, 273, 280, 284, 285, 286, 293 or 302 lacks one or several bases. Antisense oligonucleotides containing missing, substituted or inserted sequences are included. More preferably,
The sequence according to any of SEQ ID NOS: 133, 164, 244, 280, 285 or 286, or
Examples include antisense oligonucleotides containing sequences in which one or several bases have been deleted, substituted or inserted in the sequence set forth in any of SEQ ID NOs: 133, 164, 244, 280, 285 or 286. Each of these has mouse Acsl1 expression inhibitory activity. As long as the sequence is included, it is included in the antisense oligonucleotide of the present invention regardless of the length or nucleotide modification.
　本発明のアンチセンスオリゴヌクレオチドとしては、具体的には、
（Ａ－２）ＡＯＮ番号３４４～３９９のいずれかのアンチセンスヌクレオチド、又は、
ＡＯＮ番号３４４～３９９のいずれかのアンチセンスヌクレオチドにおいて、１若しくは数個の塩基が欠失、置換若しくは挿入されたアンチセンスヌクレオチド、
（Ｂ－２）ＡＯＮ番号４００～４６９、４８６～５１０のいずれかのアンチセンスヌクレオチド、又は、
ＡＯＮ番号４００～４６９、４８６～５１０のいずれかのアンチセンスヌクレオチドにおいて、１若しくは数個の塩基が欠失、置換若しくは挿入されたアンチセンスヌクレオチド、
（Ｃ－２）ＡＯＮ番号５１１、５１３～５１７、５２３～５２６、５２８～５３７、５４０～５４２、５４６、５４８～５５１、５５３、５５６～５６１、５６３～５６６、５７１～５７３、５７６、５７８～５８１、５８４、５８５、５８８、５９１～５９５、５９８、６００、６０１、６０４～６０６、６１０、６１２～６１７、６２１～６２３、６２５、６２６、６２８～６３０、６３２～６３４、６３７、６３９～６４６、６４８若しくは６５０のいずれかのアンチセンスヌクレオチド、又は、
ＡＯＮ番号５１１～６５４のいずれかのアンチセンスヌクレオチドにおいて、１若しくは数個の塩基が欠失、置換若しくは挿入されたアンチセンスヌクレオチド、
等が挙げられる。さらに好ましくは、
（Ａ－２）ＡＯＮ番号３４４～３９９のいずれかのアンチセンスヌクレオチド、又は、
ＡＯＮ番号３４４～３９９のいずれかのアンチセンスヌクレオチドにおいて、１若しくは数個の塩基が欠失、置換若しくは挿入されたアンチセンスヌクレオチド、
（Ｂ－３）ＡＯＮ番号４９４～５０５のいずれかのアンチセンスヌクレオチド、又は、
ＡＯＮ番号４９４～５０５のいずれかのアンチセンスヌクレオチドにおいて、１若しくは数個の塩基が欠失、置換若しくは挿入されたアンチセンスヌクレオチド、
（Ｃ－３）ＡＯＮ番号５３１、５４８、５６５、５８０、５８１、５９２、５９４、５９５、６０６、６１２、６１３、６３０、６３４、６４０若しくは６４５のいずれかのアンチセンスヌクレオチド、又は、
ＡＯＮ番号５３１、５４８、５６５、５８０、５８１、５９２、５９４、５９５、６０６、６１２、６１３、６３０、６３４、６４０若しくは６４５のいずれかのアンチセンスヌクレオチドにおいて、１若しくは数個の塩基が欠失、置換若しくは挿入されたアンチセンスヌクレオチド
等が挙げられる。これらはそれぞれ、ヒトＡＣＳＬ１発現抑制活性を有する。同一の配列からなる限り、ヌクレオチドの修飾の有無に関わらず、本発明のアンチセンスオリゴヌクレオチドに含まれる。
　さらに、本発明のアンチセンスオリゴヌクレオチドとしては、具体的には、
ＡＯＮ番号４７０～４８５のいずれかのアンチセンスヌクレオチド、又は、
ＡＯＮ番号４７０～４８５のいずれかのアンチセンスヌクレオチドにおいて、１若しくは数個の塩基が欠失、置換若しくは挿入されたアンチセンスヌクレオチド
等が挙げられる。さらに好ましくは
ＡＯＮ番号４７１、４７２、４７６、４８０、４８２若しくは４８３のいずれかのアンチセンスヌクレオチド、又は、
ＡＯＮ番号４７１、４７２、４７６、４８０、４８２若しくは４８３のいずれかのアンチセンスヌクレオチドにおいて、１若しくは数個の塩基が欠失、置換若しくは挿入されたアンチセンスヌクレオチド
等が挙げられる。これらはそれぞれ、マウスＡｃｓｌ１発現抑制活性を有する。同一の配列からなる限り、ヌクレオチドの修飾の有無に関わらず、本発明のアンチセンスオリゴヌクレオチドに含まれる。 As the antisense oligonucleotide of the present invention, specifically,
(A-2) an antisense nucleotide of any of AON numbers 344 to 399, or
An antisense nucleotide of any one of AON Nos. 344 to 399, wherein one or several bases are deleted, substituted or inserted;
(B-2) an antisense nucleotide of any of AON numbers 400 to 469, 486 to 510, or
An antisense nucleotide in which one or several bases are deleted, substituted or inserted in any one of the antisense nucleotides of AON Nos. 400 to 469 and 486 to 510
(C-2) AON numbers 511, 513-517, 523-526, 528-537, 540-542, 546, 548-551, 553, 556-561, 563-566, 571-573, 576, 578-581 584, 585, 588, 591 to 595, 598, 600, 601, 604 to 606, 610, 612 to 617, 621 to 623, 625, 626, 628 to 630, 632 to 634, 637, 639 to 646, 648 Or any of 650 antisense nucleotides, or
An antisense nucleotide in which one or several bases are deleted, substituted or inserted in any of the antisense nucleotides of AON Nos. 511 to 654,
Etc. More preferably,
(A-2) an antisense nucleotide of any of AON numbers 344 to 399, or
An antisense nucleotide of any one of AON Nos. 344 to 399, wherein one or several bases are deleted, substituted or inserted;
(B-3) the antisense nucleotide of any of AON numbers 494 to 505, or
An antisense nucleotide in which one or several bases have been deleted, substituted or inserted in any of the antisense nucleotides of AON Nos. 494 to 505,
(C-3) an antisense nucleotide of any one of AON numbers 531, 548, 565, 580, 581, 592, 594, 595, 606, 612, 613, 630, 634, 640 or 645, or
One or several bases are deleted in the antisense nucleotide of any one of AON numbers 531, 548, 565, 580, 581, 592, 594, 595, 606, 612, 613, 630, 634, 640 or 645, Examples include substituted or inserted antisense nucleotides. Each of these has human ACSL1 expression inhibitory activity. As long as it consists of the same sequence, it is included in the antisense oligonucleotide of the present invention regardless of whether or not the nucleotide is modified.
Furthermore, as the antisense oligonucleotide of the present invention, specifically,
An antisense nucleotide of any of AON numbers 470-485, or
Examples include antisense nucleotides in which one or several bases have been deleted, substituted, or inserted in any one of AON numbers 470 to 485. More preferably, the antisense nucleotide of any one of AON numbers 471, 472, 476, 480, 482 or 483, or
Examples include antisense nucleotides in which one or several bases are deleted, substituted or inserted in any one of the antisense nucleotides of AON Nos. 471, 472, 476, 480, 482 or 483. Each of these has mouse Acsl1 expression inhibitory activity. As long as it consists of the same sequence, it is included in the antisense oligonucleotide of the present invention regardless of whether or not the nucleotide is modified.
　なお、「１若しくは数個の塩基」とは、１～５個、好ましくは１～３個、さらに好ましくは１又は２個の塩基を意味している。欠失、置換、挿入又は付加によっても、標的遺伝子（例えば、ＡＣＳＬ１）の発現抑制作用を有する限り、本発明のアンチセンスオリゴヌクレオチドに包含される。 Note that “one or several bases” means 1 to 5, preferably 1 to 3, more preferably 1 or 2 bases. The antisense oligonucleotide of the present invention includes any deletion, substitution, insertion or addition as long as it has an action of suppressing the expression of the target gene (for example, ACSL1).
　本発明のアンチセンスオリゴヌクレオチドは、ＡＣＳＬ１発現抑制活性のみならず、医薬としての有用性を備えており、下記いずれか、あるいは全ての優れた特徴を有している。
ａ）ＣＹＰ酵素（例えば、ＣＹＰ１Ａ２、ＣＹＰ２Ｃ９、ＣＹＰ２Ｃ１９、ＣＹＰ２Ｄ６、ＣＹＰ３Ａ４等）に対する阻害作用が弱い。
ｂ）高いバイオアベイラビリティー、適度なクリアランス等良好な薬物動態を示す。
ｃ）代謝安定性が高い。
ｄ）変異原性を有さない。
ｅ）心血管系のリスクが低い。
ｆ）高い溶解性を示す。
ｇ）ＡＣＳＬ１特異性を示す。
ｈ）肝臓毒性を示さない。 The antisense oligonucleotide of the present invention has not only ACSL1 expression-suppressing activity but also usefulness as a medicine, and has any or all of the following excellent features.
a) The inhibitory effect on CYP enzymes (for example, CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, etc.) is weak.
b) Good pharmacokinetics such as high bioavailability and moderate clearance.
c) High metabolic stability.
d) Not mutagenic.
e) Low cardiovascular risk.
f) High solubility.
g) Shows ACSL1 specificity.
h) Does not show liver toxicity.
　本発明のアンチセンスオリゴヌクレオチドにおいて、ヌクレオチドは修飾されていてもよい。適切な修飾を施したアンチセンスオリゴヌクレオチドは、無修飾のアンチセンスオリゴヌクレオチドと比較し、下記いずれか、あるいは全ての特徴を有している。
ａ）　標的遺伝子との親和性が高い。
ｂ）　ヌクレアーゼに対する抵抗性が高い。
ｃ）　薬物動態が改善する。
ｄ）　組織移行性が高くなる。
　よって、修飾されたアンチセンスオリゴヌクレオチドは、無修飾のアンチセンスオリゴヌクレオチドと比較して、生体内で分解されにくくなり、より安定して標的遺伝子の発現を阻害できる。 In the antisense oligonucleotide of the present invention, the nucleotide may be modified. Antisense oligonucleotides with appropriate modifications have any or all of the following characteristics compared to unmodified antisense oligonucleotides.
a) High affinity with the target gene.
b) High resistance to nucleases.
c) Improved pharmacokinetics.
d) Increases organizational transferability.
Therefore, the modified antisense oligonucleotide is less likely to be degraded in vivo than the unmodified antisense oligonucleotide, and can inhibit the expression of the target gene more stably.
　当該分野で公知のヌクレオチドの修飾であれば、いずれも本発明のアンチセンスオリゴヌクレオチドに利用可能である。ヌクレオチドの修飾としては、リン酸修飾、核酸塩基修飾、糖修飾が知られている。
　リン酸修飾としては、例えば、天然の核酸が有するリン酸ジエステル結合、Ｓ－オリゴ（ホスホロチオエート）、Ｄ－オリゴ（ホスホジエステル）、Ｍ－オリゴ（メチルフォスフォネイト）、ボラノホスフェート等が挙げられる。
　核酸塩基修飾としては、例えば、５－メチルシトシン、５－ヒドロキシメチルシトシン、５－プロピニルシトシン等が挙げられる。
　糖修飾としては、例えば、２’－Ｏ－ＣＨ２－ＣＨ２－Ｏ－ＣＨ３（２’ＭＯＥ）、後述する実施例１に記載の式（ａ）で示されるＬＮＡ（Ｌｏｃｋｅｄ　ｎｕｃｌｅｉｃ　ａｃｉｄ）、式（ｂ）で示される２’‐ＯＭｅ、式（ｃ）で示されるアミドＢＮＡ（Ｂｒｉｄｇｅｄ　ｎｕｃｌｅｉｃ　ａｃｉｄ、ＡｍＮＡ）等が挙げられる。 Any nucleotide modification known in the art can be used for the antisense oligonucleotide of the present invention. Known modifications of nucleotides include phosphate modification, nucleobase modification, and sugar modification.
Examples of phosphoric acid modifications include phosphodiester bonds, S-oligos (phosphorothioates), D-oligos (phosphodiesters), M-oligos (methyl phosphonates), boranophosphates, etc. possessed by natural nucleic acids. .
Examples of the nucleobase modification include 5-methylcytosine, 5-hydroxymethylcytosine, 5-propynylcytosine and the like.
Examples of the sugar modification include 2′-O—CH 2 —CH 2 —O—CH 3 (2′MOE), LNA (Locked Nucleic Acid) represented by Formula (a) described in Example 1 described later, Examples thereof include 2′-OMe represented by the formula (b), amide BNA (Bridged nucleic acid, AmNA) represented by the formula (c), and the like.
　当該分野で公知のヌクレオチドの修飾及び修飾方法については、例えば、以下の特許文献にも開示されている。
国際公開第９８／３９３５２号、国際公開第９９／０１４２２６号、国際公開第２０００／０５６７４８、国際公開第２００５／０２１５７０号、国際公開第２００３／０６８７９５号、国際公開第２０１１／０５２４３６号、国際公開第２００４／０１６７４９号、国際公開第２００５／０８３１２４号、国際公開第２００７／１４３３１５号、国際公開第２００９／０７１６８０号、国際公開第２０１４／１１２４６３、国際出願ＰＣＴ／ＪＰ２０１４／０５３５８０等。 Nucleotide modification and modification methods known in the art are also disclosed in, for example, the following patent documents.
International Publication No. 98/39352, International Publication No. 99/014226, International Publication No. 2000/056748, International Publication No. 2005/021570, International Publication No. 2003/066875, International Publication No. 2011/052436, International Publication No. 2004/016749, International Publication No. 2005/083124, International Publication No. 2007/143315, International Publication No. 2009/071680, International Publication No. 2014/112463, International Application PCT / JP2014 / 053580, and the like.
　以下に、本発明のアンチセンスオリゴヌクレオチドに利用可能な修飾を具体的に例示するが、該修飾はこれらにより限定されるものではない。
（１）Ｓ－オリゴ（ホスホロチオエート）
　ヌクレオシド間のホスホジエステル結合のリン酸基部の酸素原子を硫黄原子で置換する。該修飾は公知の方法に従って、オリゴヌクレオチドに取り込まれる。該修飾をオリゴヌクレオチド中に１若しくは複数もつアンチセンスオリゴヌクレオチドをＳ－オリゴ型（ホスホロチオエート型）という。
（２）ＬＮＡ（Ｌｏｃｋｅｄ　ｎｕｃｌｅｉｃ　ａｃｉｄ）
　ヌクレオチドの糖環の４’炭素原子から適切な架橋を介して２’－ヒドロキシル基が結合して二環式糖部分が形成される。好ましい結合は、２’酸素原子と４’炭素原子とを架橋するメチレン（－ＣＨ２－）基である。ＬＮＡの具体例及びその調製方法は、国際公開第９８／３９３５２号、国際公開第２００３／０６８７９５号、国際公開第２００５／０２１５７０号等に記載されている。
（３）アミドＢＮＡ（Ｂｒｉｄｇｅｄ　ｎｕｃｌｅｉｃ　ａｃｉｄ、ＡｍＮＡ）
　ヌクレオチドの糖環の２’のアミノ基と４’から伸長したカルボニル基との間にアミド結合が形成されることにより、二環式糖部分が形成される。アミドＢＮＡの具体例及びその調製方法は、国際公開第２０１１／０５２４３６号に記載されている。
（４）ヌクレオチドの糖の２’位置の修飾
　ヌクレオチドの糖の２’位置のＯＨを－Ｏ－アルキル（例えば、－Ｏ－メチル、－Ｏ－エチル、－Ｏ－メトキシエチル等）、又はＦで置換する。該修飾は公知の方法に従って、オリゴヌクレオチドに取り込まれる。 Hereinafter, modifications that can be used for the antisense oligonucleotide of the present invention are specifically exemplified, but the modifications are not limited thereto.
(1) S-oligo (phosphorothioate)
The oxygen atom of the phosphate group of the phosphodiester bond between nucleosides is replaced with a sulfur atom. The modification is incorporated into the oligonucleotide according to known methods. An antisense oligonucleotide having one or more such modifications in the oligonucleotide is referred to as an S-oligo type (phosphorothioate type).
(2) LNA (Locked Nucleic Acid)
A 2'-hydroxyl group is attached from the 4 'carbon atom of the sugar ring of the nucleotide via an appropriate bridge to form a bicyclic sugar moiety. A preferred bond is a methylene (—CH 2 —) group that bridges the 2 ′ oxygen atom and the 4 ′ carbon atom. Specific examples of LNA and preparation methods thereof are described in International Publication No. 98/39352, International Publication No. 2003/068795, International Publication No. 2005/021570, and the like.
(3) Amide BNA (Bridged nucleic acid, AmNA)
An amide bond is formed between the 2 ′ amino group of the sugar ring of the nucleotide and the carbonyl group extended from 4 ′ to form a bicyclic sugar moiety. Specific examples of amide BNA and the preparation method thereof are described in WO 2011/052436.
(4) Modification of the 2′-position of a nucleotide sugar Replace. The modification is incorporated into the oligonucleotide according to known methods.
　本発明のアンチセンスオリゴヌクレオチドは、ギャップマーであることが好ましい。ギャップマーとは、中心領域（“ギャップ”）と該中心領域の両側の領域、ウイング（５’側の“５’ウイング”又は３’側の“３’ウイング”）を含み、各ウイングに少なくとも１つの修飾ヌクレオチドを含むオリゴヌクレオチドを意味する。修飾ヌクレオチドの修飾は、リン酸修飾、塩基修飾、糖修飾のいずれであってもよい。一方のウイング内の修飾の種類、数、位置は他方のウイングにおける修飾の種類、数、位置と同じであっても異なっていてもよい。 The antisense oligonucleotide of the present invention is preferably a gapmer. A gapmer includes a central region (“gap”) and regions on both sides of the central region, wings (“5 ′ wing” on the 5 ′ side or “3 ′ wing” on the 3 ′ side), and each wing has at least It means an oligonucleotide containing one modified nucleotide. The modification of the modified nucleotide may be any of phosphate modification, base modification, and sugar modification. The type, number, and position of the modification in one wing may be the same as or different from the type, number, and position of the modification in the other wing.
　好ましくは、本発明のアンチセンスは、「４’位と２’位との間に架橋構造を有する糖修飾ヌクレオシドを１以上含有」する。好ましくは、ギャップマーである。“５’ウイング”及び ／又は“３’ウイング”に、４’位と２’位との間に架橋構造を有する糖修飾ヌクレオシドを１以上、好ましくは、１～５、さらに好ましくは、２～３含有する。また、さらに、リン酸修飾、塩基修飾、他の糖修飾を含んでいてもよい。 Preferably, the antisense of the present invention contains “one or more sugar-modified nucleosides having a bridging structure between the 4′-position and the 2′-position”. Gapmer is preferable. “5 ′ wing” and cocoon / or “3 ′ wing” include one or more sugar-modified nucleosides having a cross-linked structure between the 4′-position and the 2′-position, preferably 1-5, more preferably 2- 3 contained. Furthermore, phosphate modification, base modification, and other sugar modifications may be included.
　「４’位と２’位との間に架橋構造を有する糖修飾ヌクレオシド」とは、糖の４’位と２’位との間に架橋構造を有する修飾を含む公知のヌクレオシドであれば、いずれでもよい。また、さらに、塩基修飾、他の糖修飾を含んでいてもよい。 The “sugar-modified nucleoside having a crosslinking structure between the 4′-position and the 2′-position” is a known nucleoside containing a modification having a crosslinking structure between the 4′-position and the 2′-position of the sugar, Either is acceptable. Furthermore, base modification and other sugar modifications may be included.
　該架橋構造として、具体的には、以下が挙げられる。
　４’－（ＣＲ２Ｒ３）ｍ－Ｏ－２’、４’－（ＣＲ２Ｒ３）ｍ－ＮＲ１－Ｏ－２’、４’－（ＣＲ２Ｒ３）ｎ-ＣＯ－ＮＲ１－２’又は４’ －（ＣＲ２Ｒ３）ｎ-ＣＯ－ＮＲ１－Ｘ－２’であり、
ここで、
Ｘは、酸素原子、硫黄原子、アミノ又はＣＲ２Ｒ３であり、
Ｒ１は、水素原子、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル、置換若しくは非置換のアルキニル、置換若しくは非置換の芳香族炭素環式基、置換若しくは非置換の非芳香族炭素環式基、置換若しくは非置換の芳香族複素環式基、置換若しくは非置換の非芳香族複素環式基、置換若しくは非置換の芳香族炭素環アルキル、置換若しくは非置換の非芳香族炭素環アルキル、置換若しくは非置換の芳香族複素環アルキル又は置換若しくは非置換の非芳香族複素環アルキルであり、
Ｒ２は、それぞれ独立して、水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキ
ル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルであり、
Ｒ３は、それぞれ独立して、水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルであり、
ｍは、１～３であり、
ｎは、０～３である。 Specific examples of the crosslinked structure include the following.
4 ′-(CR 2 R 3 ) m —O-2 ′, 4 ′-(CR 2 R 3 ) m —NR 1 —O-2 ′, 4 ′-(CR 2 R 3 ) n —CO—NR 1 -2 'or 4'-(CR 2 R 3 ) n -CO-NR 1 -X-2 ',
here,
X is an oxygen atom, a sulfur atom, amino or CR 2 R 3 ,
R 1 is a hydrogen atom, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aromatic carbocyclic group, substituted or unsubstituted non-aromatic carbocycle Group, substituted or unsubstituted aromatic heterocyclic group, substituted or unsubstituted non-aromatic heterocyclic group, substituted or unsubstituted aromatic carbocyclic alkyl, substituted or unsubstituted non-aromatic carbocyclic alkyl Substituted or unsubstituted aromatic heterocyclic alkyl or substituted or unsubstituted non-aromatic heterocyclic alkyl,
Each R 2 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl;
Each R 3 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl;
m is 1 to 3,
n is 0-3.
　Ｒ１は、好ましくは、水素原子、アルキル、アルケニル、アルキニル、芳香族炭素環式基、非芳香族炭素環式基、芳香族複素環式基、非芳香族複素環式基、芳香族炭素環アルキル、非芳香族炭素環アルキル、芳香族複素環アルキル又は非芳香族複素環アルキルであり、α群から選択される任意の置換基を１以上有していてもよい。
　α群は、水酸基、アルキル、アルキルオキシ、メルカプト、アルキルチオ、アミノ、アルキルアミノ又はハロゲンである。 R 1 is preferably a hydrogen atom, alkyl, alkenyl, alkynyl, aromatic carbocyclic group, non-aromatic carbocyclic group, aromatic heterocyclic group, non-aromatic heterocyclic group, aromatic carbocycle It is alkyl, non-aromatic carbocyclic alkyl, aromatic heterocyclic alkyl or non-aromatic heterocyclic alkyl, and may have one or more arbitrary substituents selected from α group.
The α group is a hydroxyl group, alkyl, alkyloxy, mercapto, alkylthio, amino, alkylamino or halogen.
　Ｒ２及びＲ３は、好ましくは、水素原子である。
　ｍは、好ましくは１又は２である。
　ｎは、好ましくは０又は１である。 R 2 and R 3 are preferably a hydrogen atom.
m is preferably 1 or 2.
n is preferably 0 or 1.
　該架橋構造として、好ましくは、４’－（ＣＲ２Ｒ３）ｍ－Ｏ－２’、又は、４’－（ＣＲ２Ｒ３）ｎ-ＣＯ－ＮＲ１－２’であり、
ここで、
Ｒ１は、水素原子、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルであり、
Ｒ２は、それぞれ独立して、水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルであり、
Ｒ３は、それぞれ独立して、水素原子、ハロゲン、シアノ、置換若しくは非置換のアルキル、置換若しくは非置換のアルケニル又は置換若しくは非置換のアルキニルであり、
ｍは、１～３であり、
ｎは、０～３である。 The cross-linked structure is preferably 4 ′-(CR 2 R 3 ) m —O-2 ′ or 4 ′-(CR 2 R 3 ) n —CO—NR 1 −2 ′.
here,
R 1 is a hydrogen atom, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl or substituted or unsubstituted alkynyl,
Each R 2 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl;
Each R 3 is independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl;
m is 1 to 3,
n is 0-3.
　該架橋構造として、特に好ましくは、４’－ＣＨ２－Ｏ－２’又は４’－ＣＯ－ＮＲ１－２’（Ｒ１は、水素原子又はアルキルである）である。 The cross-linked structure is particularly preferably 4′-CH 2 —O-2 ′ or 4′-CO—NR 1 -2 ′ (R 1 is a hydrogen atom or alkyl).
　「ハロゲン」とは、フッ素原子、塩素原子、臭素原子、及びヨウ素原子を包含する。特にフッ素原子、及び塩素原子が好ましい。 “Halogen” includes fluorine atom, chlorine atom, bromine atom and iodine atom. In particular, a fluorine atom and a chlorine atom are preferable.
　「アルキル」とは、炭素数１～１５、好ましくは炭素数１～１０、より好ましくは炭素数１～６、さらに好ましくは炭素数１～４の直鎖又は分枝状の炭化水素基を包含する。例えば、メチル、エチル、ｎ－プロピル、イソプロピル、ｎ－ブチル、イソブチル、ｓｅｃ－ブチル、ｔｅｒｔ－ブチル、ｎ－ペンチル、イソペンチル、ネオペンチル、ｎ－ヘキシル、イソヘキシル、ｎ－へプチル、イソヘプチル、ｎ－オクチル、イソオクチル、ｎ－ノニル、ｎ－デシル等が挙げられる。
　「アルキル」の好ましい態様として、メチル、エチル、ｎ－プロピル、イソプロピル、ｎ－ブチル、イソブチル、ｓｅｃ－ブチル、ｔｅｒｔ－ブチル、ｎ－ペンチルが挙げられる。さらに好ましい態様として、メチル、エチル、ｎ－プロピル、イソプロピル、ｔｅｒｔ－ブチルが挙げられる。 “Alkyl” includes straight or branched hydrocarbon groups having 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, and still more preferably 1 to 4 carbon atoms. To do. For example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl , Isooctyl, n-nonyl, n-decyl and the like.
Preferred embodiments of “alkyl” include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and n-pentyl. Further preferred examples include methyl, ethyl, n-propyl, isopropyl and tert-butyl.
　「アルケニル」とは、任意の位置に１以上の二重結合を有する、炭素数２～１５、好ましくは炭素数２～１０、より好ましくは炭素数２～６、さらに好ましくは炭素数２～４の直鎖又は分枝状の炭化水素基を包含する。例えば、ビニル、アリル、プロペニル、イソプロペニル、ブテニル、イソブテニル、プレニル、ブタジエニル、ペンテニル、イソペンテニル、ペンタジエニル、ヘキセニル、イソヘキセニル、ヘキサジエニル、ヘプテニル、オクテニル、ノネニル、デセニル、ウンデセニル、ドデセニル、トリデセニル、テトラデセニル、ペンタデセニル等が挙げられる。
　「アルケニル」の好ましい態様として、ビニル、アリル、プロペニル、イソプロペニル、ブテニルが挙げられる。 “Alkenyl” has 2 to 15 carbon atoms, preferably 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and further preferably 2 to 4 carbon atoms, having one or more double bonds at any position. These linear or branched hydrocarbon groups are included. For example, vinyl, allyl, propenyl, isopropenyl, butenyl, isobutenyl, prenyl, butadienyl, pentenyl, isopentenyl, pentadienyl, hexenyl, isohexenyl, hexadienyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, decenyl, tridecenyl, decenyl Etc.
Preferred embodiments of “alkenyl” include vinyl, allyl, propenyl, isopropenyl and butenyl.
　「アルキニル」とは、任意の位置に１以上の三重結合を有する、炭素数２～１０、好ましくは炭素数２～８、さらに好ましくは炭素数２～６、さらに好ましくは炭素数２～４の直鎖又は分枝状の炭化水素基を包含する。例えば、エチニル、プロピニル、ブチニル、ペンチニル、ヘキシニル、ヘプチニル、オクチニル、ノニニル、デシニル等を包含する。これらはさらに任意の位置に二重結合を有していてもよい。
　「アルキニル」の好ましい態様として、エチニル、プロピニル、ブチニル、ペンチニルが挙げられる。 “Alkynyl” has 2 to 10 carbon atoms, preferably 2 to 8 carbon atoms, more preferably 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms, having one or more triple bonds at any position. Includes straight chain or branched hydrocarbon groups. Examples include ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl and the like. These may further have a double bond at an arbitrary position.
Preferred embodiments of “alkynyl” include ethynyl, propynyl, butynyl and pentynyl.
　「芳香族炭素環式基」とは、単環又は２環以上の、環状芳香族炭化水素基を意味する。例えば、フェニル、ナフチル、アントリル、フェナントリル等が挙げられる。
　「芳香族炭素環式基」の好ましい態様として、フェニルが挙げられる。 “Aromatic carbocyclic group” means a monocyclic or bicyclic or more cyclic aromatic hydrocarbon group. For example, phenyl, naphthyl, anthryl, phenanthryl and the like can be mentioned.
A preferred embodiment of the “aromatic carbocyclic group” includes phenyl.
　「非芳香族炭素環式基」とは、単環又は２環以上の、環状飽和炭化水素基又は環状非芳香族不飽和炭化水素基を意味する。２環以上の非芳香族炭素環式基は、単環又は２環以上の非芳香族炭素環式基に、上記「芳香族炭素環式基」における環が縮合したものも包含する。
　さらに、「非芳香族炭素環式基」は、以下のように架橋している基、又はスピロ環を形成する基も包含する。
Figure JPOXMLDOC01-appb-C000001
　単環の非芳香族炭素環式基としては、炭素数３～１６が好ましく、より好ましくは炭素数３～１２、さらに好ましくは炭素数４～８である。例えば、シクロプロピル、シクロブチル、シクロペンチル、シクロヘキシル、シクロヘプチル、シクロオクチル、シクロノニル、シクロデシル、シクロプロペニル、シクロブテニル、シクロペンテニル、シクロヘキセニル、シクロヘプテニル、シクロヘキサジエニル等が挙げられる。
　２環以上の非芳香族炭素環式基としては、例えば、インダニル、インデニル、アセナフチル、テトラヒドロナフチル、フルオレニル等が挙げられる。 The “non-aromatic carbocyclic group” means a cyclic saturated hydrocarbon group or a cyclic non-aromatic unsaturated hydrocarbon group having one or more rings. The non-aromatic carbocyclic group having two or more rings includes a monocyclic ring or a non-aromatic carbocyclic group having two or more rings condensed with the ring in the above “aromatic carbocyclic group”.
Furthermore, the “non-aromatic carbocyclic group” includes a group which forms a bridge or a spiro ring as described below.
Figure JPOXMLDOC01-appb-C000001
The monocyclic non-aromatic carbocyclic group preferably has 3 to 16 carbon atoms, more preferably 3 to 12 carbon atoms, and still more preferably 4 to 8 carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclohexadienyl, and the like.
Examples of the two or more non-aromatic carbocyclic groups include indanyl, indenyl, acenaphthyl, tetrahydronaphthyl, fluorenyl and the like.
　「芳香族複素環式基」とは、Ｏ、Ｓ及びＮから任意に選択される同一又は異なるヘテロ原子を環内に１以上有する、単環又は２環以上の、芳香族環式基を意味する。
　２環以上の芳香族複素環式基は、単環又は２環以上の芳香族複素環式基に、上記「芳香族炭素環式基」における環が縮合したものも包含する。
　単環の芳香族複素環式基としては、５～８員が好ましく、より好ましくは５員又は６員である。例えば、ピロリル、イミダゾリル、ピラゾリル、ピリジル、ピリダジニル、ピリミジニル、ピラジニル、トリアゾリル、トリアジニル、テトラゾリル、フリル、チエニル、イソオキサゾリル、オキサゾリル、オキサジアゾリル、イソチアゾリル、チアゾリル、チアジアゾリル等が挙げられる。
　２環の芳香族複素環式基としては、例えば、インドリル、イソインドリル、インダゾリル、インドリジニル、キノリニル、イソキノリニル、シンノリニル、フタラジニル、キナゾリニル、ナフチリジニル、キノキサリニル、プリニル、プテリジニル、ベンズイミダゾリル、ベンズイソオキサゾリル、ベンズオキサゾリル、ベンズオキサジアゾリル、ベンズイソチアゾリル、ベンゾチアゾリル、ベンゾチアジアゾリル、ベンゾフリル、イソベンゾフリル、ベンゾチエニル、ベンゾトリアゾリル、イミダゾピリジル、トリアゾロピリジル、イミダゾチアゾリル、ピラジノピリダジニル、オキサゾロピリジル、チアゾロピリジル等が挙げられる。
　３環以上の芳香族複素環式基としては、例えば、カルバゾリル、アクリジニル、キサンテニル、フェノチアジニル、フェノキサチイニル、フェノキサジニル、ジベンゾフリル等が挙げられる。 “Aromatic heterocyclic group” means a monocyclic or bicyclic or more aromatic cyclic group having one or more heteroatoms arbitrarily selected from O, S and N in the ring. To do.
The aromatic heterocyclic group having two or more rings includes those obtained by condensing a ring in the above “aromatic carbocyclic group” to a monocyclic or two or more aromatic heterocyclic group.
The monocyclic aromatic heterocyclic group is preferably 5 to 8 members, more preferably 5 or 6 members. Examples include pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazolyl, triazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, oxazolyl, oxadiazolyl, isothiazolyl, thiazolyl, thiadiazolyl and the like.
Examples of the bicyclic aromatic heterocyclic group include indolyl, isoindolyl, indazolyl, indolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, naphthyridinyl, quinoxalinyl, purinyl, pteridinyl, benzimidazolyl, benzisoxazolyl, benzisoxazolyl, Oxazolyl, benzoxiadiazolyl, benzisothiazolyl, benzothiazolyl, benzothiadiazolyl, benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl, imidazopyridyl, triazolopyridyl, imidazothiazolyl, pyrazinopyr Dazinyl, oxazolopyridyl, thiazolopyridyl and the like can be mentioned.
Examples of the aromatic heterocyclic group having 3 or more rings include carbazolyl, acridinyl, xanthenyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, dibenzofuryl and the like.
　「非芳香族複素環式基」とは、Ｏ、Ｓ及びＮから任意に選択される同一又は異なるヘテロ原子を環内に１以上有する、単環又は２環以上の、環状非芳香族環式基を意味する。
　２環以上の非芳香族複素環式基は、単環又は２環以上の非芳香族複素環式基に、上記「芳香族炭素環式基」、「非芳香族炭素環式基」、及び／又は「芳香族複素環式基」におけるそれぞれの環が縮合したものも包含する。
　さらに、「非芳香族複素環式基」は、以下のように架橋している基、又はスピロ環を形成する基も包含する。
Figure JPOXMLDOC01-appb-C000002
　単環の非芳香族複素環式基としては、３～８員が好ましく、より好ましくは５員又は６員である。例えば、ジオキサニル、チイラニル、オキシラニル、オキセタニル、オキサチオラニル、アゼチジニル、チアニル、チアゾリジニル、ピロリジニル、ピロリニル、イミダゾリジニル、イミダゾリニル、ピラゾリジニル、ピラゾリニル、ピペリジル、ピペラジニル、モルホリニル、モルホリノ、チオモルホリニル、チオモルホリノ、ジヒドロピリジル、テトラヒドロピリジル、テトラヒドロフリル、テトラヒドロピラニル、ジヒドロチアゾリル、テトラヒドロチアゾリル、テトラヒドロイソチアゾリル、ジヒドロオキサジニル、ヘキサヒドロアゼピニル、テトラヒドロジアゼピニル、テトラヒドロピリダジニル、ヘキサヒドロピリミジニル、ジオキソラニル、ジオキサジニル、アジリジニル、ジオキソリニル、オキセパニル、チオラニル、チイニル、チアジニル等が挙げられる。
　２環以上の非芳香族複素環式基としては、例えば、インドリニル、イソインドリニル、クロマニル、イソクロマニル等が挙げられる。 The “non-aromatic heterocyclic group” is a monocyclic or bicyclic or more cyclic non-aromatic cyclic group having one or more of the same or different heteroatoms arbitrarily selected from O, S and N in the ring Means group.
The non-aromatic heterocyclic group having two or more rings includes the above-mentioned “aromatic carbocyclic group”, “non-aromatic carbocyclic group”, and monocyclic or two or more non-aromatic heterocyclic groups, and Also included are those in which each ring in the “aromatic heterocyclic group” is condensed.
Furthermore, the “non-aromatic heterocyclic group” also includes a group that forms a bridge or a spiro ring as described below.
Figure JPOXMLDOC01-appb-C000002
The monocyclic non-aromatic heterocyclic group is preferably 3 to 8 members, more preferably 5 or 6 members. For example, dioxanyl, thiranyl, oxiranyl, oxetanyl, oxathiolanyl, azetidinyl, thianyl, thiazolidinyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidyl, piperazinyl, morpholinyl, morpholino, thiomorpholinyl, morpholino, thiomorpholinyl, morpholino, thiomorpholinyl Furyl, tetrahydropyranyl, dihydrothiazolyl, tetrahydrothiazolyl, tetrahydroisothiazolyl, dihydrooxazinyl, hexahydroazepinyl, tetrahydrodiazepinyl, tetrahydropyridazinyl, hexahydropyrimidinyl, dioxolanyl, dioxazinyl Aziridinyl, dioxolinyl, oxepanyl, thiolanyl, thii Le, triazinyl, and the like.
Examples of the non-aromatic heterocyclic group having two or more rings include indolinyl, isoindolinyl, chromanyl, isochromanyl and the like.
　「アルキルオキシ」とは、上記「アルキル」が酸素原子に結合した基を意味する。例えば、メトキシ、エトキシ、ｎ－プロピルオキシ、イソプロピルオキシ、ｎ－ブチルオキシ、ｔｅｒｔ－ブチルオキシ、イソブチルオキシ、ｓｅｃ－ブチルオキシ、ペンチルオキシ、イソペンチルオキシ、へキシルオキシ等が挙げられる。
　「アルキルオキシ」の好ましい態様として、メトキシ、エトキシ、ｎ－プロピルオキシ、イソプロピルオキシ、ｔｅｒｔ－ブチルオキシが挙げられる。 “Alkyloxy” means a group in which the above “alkyl” is bonded to an oxygen atom. Examples thereof include methoxy, ethoxy, n-propyloxy, isopropyloxy, n-butyloxy, tert-butyloxy, isobutyloxy, sec-butyloxy, pentyloxy, isopentyloxy, hexyloxy and the like.
Preferable embodiments of “alkyloxy” include methoxy, ethoxy, n-propyloxy, isopropyloxy, tert-butyloxy.
　「アルキルアミノ」には、モノアルキルアミノとジアルキルアミノが含まれる。
　「モノアルキルアミノ」とは、上記「アルキル」がアミノ基の窒素原子と結合している水素原子１個と置き換わった基を意味する。例えば、メチルアミノ、エチルアミノ、イソプロピルアミノ等が挙げられる。好ましくは、メチルアミノ、エチルアミノが挙げられる。
　「ジアルキルアミノ」とは、上記「アルキル」がアミノ基の窒素原子と結合している水素原子２個と置き換わった基を意味する。２個のアルキル基は、同一でも異なっていてもよい。例えば、ジメチルアミノ、ジエチルアミノ、Ｎ，Ｎ－ジイソプロピルアミノ、Ｎ－メチル－Ｎ－エチルアミノ、Ｎ－イソプロピル－Ｎ－エチルアミノ等が挙げられる。好ましくは、ジメチルアミノ、ジエチルアミノが挙げられる。 “Alkylamino” includes monoalkylamino and dialkylamino.
“Monoalkylamino” means a group in which the above “alkyl” is replaced with one hydrogen atom bonded to the nitrogen atom of the amino group. For example, methylamino, ethylamino, isopropylamino and the like can be mentioned. Preferably, methylamino and ethylamino are used.
“Dialkylamino” means a group in which the above “alkyl” is replaced with two hydrogen atoms bonded to the nitrogen atom of the amino group. Two alkyl groups may be the same or different. Examples include dimethylamino, diethylamino, N, N-diisopropylamino, N-methyl-N-ethylamino, N-isopropyl-N-ethylamino and the like. Preferable examples include dimethylamino and diethylamino.
　「アルキルチオ」とは、上記「アルキル」が硫黄原子に結合した基を意味する。 “Alkylthio” means a group in which the above “alkyl” is bonded to a sulfur atom.
　「置換若しくは非置換のアルキル」、「置換若しくは非置換のアルケニル」、「置換若しくは非置換のアルキニル」の置換基としては、次の置換基が挙げられる。任意の位置の炭素原子が次の置換基から選択される１以上の基と結合していてもよい。
　置換基：ハロゲン、ヒドロキシ、カルボキシ、アミノ、イミノ、ヒドロキシアミノ、ヒドロキシイミノ、ホルミル、ホルミルオキシ、カルバモイル、スルファモイル、スルファニル、スルフィノ、スルホ、チオホルミル、チオカルボキシ、ジチオカルボキシ、チオカルバモイル、シアノ、ニトロ、ニトロソ、アジド、ヒドラジノ、ウレイド、アミジノ、グアニジノ、トリアルキルシリル、アルキルオキシ、アルケニルオキシ、アルキニルオキシ、ハロアルキルオキシ、アルキルカルボニル、アルケニルカルボニル、アルキニルカルボニル、モノアルキルアミノ、ジアルキルアミノ、アルキルスルホニル、アルケニルスルホニル、アルキニルスルホニル、モノアルキルカルボニルアミノ、ジアルキルカルボニルアミノ、モノアルキルスルホニルアミノ、ジアルキルスルホニルアミノ、アルキルイミノ、アルケニルイミノ、アルキニルイミノ、アルキルカルボニルイミノ、アルケニルカルボニルイミノ、アルキニルカルボニルイミノ、アルキルオキシイミノ、アルケニルオキシイミノ、アルキニルオキシイミノ、アルキルカルボニルオキシ、アルケニルカルボニルオキシ、アルキニルカルボニルオキシ、アルキルオキシカルボニル、アルケニルオキシカルボニル、アルキニルオキシカルボニル、アルキルスルファニル、アルケニルスルファニル、アルキニルスルファニル、アルキルスルフィニル、アルケニルスルフィニル、アルキニルスルフィニル、モノアルキルカルバモイル、ジアルキルカルバモイル、モノアルキルスルファモイル、ジアルキルスルファモイル、芳香族炭素環式基、非芳香族炭素環式基、芳香族複素環式基、非芳香族複素環式基、芳香族炭素環オキシ、非芳香族炭素環オキシ、芳香族複素環オキシ、非芳香族複素環オキシ、芳香族炭素環カルボニル、非芳香族炭素環カルボニル、芳香族複素環カルボニル、非芳香族複素環カルボニル、芳香族炭素環オキシカルボニル、非芳香族炭素環オキシカルボニル、芳香族複素環オキシカルボニル、非芳香族複素環オキシカルボニル、芳香族炭素環アルキルオキシ、非芳香族炭素環アルキルオキシ、芳香族複素環アルキルオキシ、非芳香族複素環アルキルオキシ、芳香族炭素環アルキルオキシカルボニル、非芳香族炭素環アルキルオキシカルボニル、芳香族複素環アルキルオキシカルボニル、非芳香族複素環アルキルオキシカルボニル、芳香族炭素環アルキルアミノ、非芳香族炭素環アルキルアミノ、芳香族複素環アルキルアミノ、非芳香族複素環アルキルアミノ、芳香族炭素環スルファニル、非芳香族炭素環スルファニル、芳香族複素環スルファニル、非芳香族複素環スルファニル、非芳香族炭素環スルホニル、芳香族炭素環スルホニル、芳香族複素環スルホニル、及び非芳香族複素環スルホニル。 Examples of the substituent of “substituted or unsubstituted alkyl”, “substituted or unsubstituted alkenyl”, and “substituted or unsubstituted alkynyl” include the following substituents. The carbon atom at any position may be bonded to one or more groups selected from the following substituents.
Substituents: halogen, hydroxy, carboxy, amino, imino, hydroxyamino, hydroxyimino, formyl, formyloxy, carbamoyl, sulfamoyl, sulfanyl, sulfino, sulfo, thioformyl, thiocarboxy, dithiocarboxy, thiocarbamoyl, cyano, nitro, nitroso , Azide, hydrazino, ureido, amidino, guanidino, trialkylsilyl, alkyloxy, alkenyloxy, alkynyloxy, haloalkyloxy, alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, monoalkylamino, dialkylamino, alkylsulfonyl, alkenylsulfonyl, alkynyl Sulfonyl, monoalkylcarbonylamino, dialkylcarbonylamino, monoalkylsulfonyl Mino, dialkylsulfonylamino, alkylimino, alkenylimino, alkynylimino, alkylcarbonylimino, alkenylcarbonylimino, alkynylcarbonylimino, alkyloxyimino, alkenyloxyimino, alkynyloxyimino, alkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy Alkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, alkylsulfanyl, alkenylsulfanyl, alkynylsulfanyl, alkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, monoalkylcarbamoyl, dialkylcarbamoyl, monoalkylsulfamoyl, dialkylsulfamoyl, aromatic Group carbocyclic group, Aromatic carbocyclic group, aromatic heterocyclic group, non-aromatic heterocyclic group, aromatic carbocyclic oxy, non-aromatic carbocyclic oxy, aromatic heterocyclic oxy, non-aromatic heterocyclic oxy, aromatic Carbocyclic carbonyl, non-aromatic carbocyclic carbonyl, aromatic heterocyclic carbonyl, non-aromatic heterocyclic carbonyl, aromatic carbocyclic oxycarbonyl, non-aromatic carbocyclic oxycarbonyl, aromatic heterocyclic oxycarbonyl, non-aromatic hetero Ring oxycarbonyl, aromatic carbocyclic alkyloxy, non-aromatic carbocyclic alkyloxy, aromatic heterocyclic alkyloxy, non-aromatic heterocyclic alkyloxy, aromatic carbocyclic alkyloxycarbonyl, non-aromatic carbocyclic alkyloxycarbonyl , Aromatic heterocyclic alkyloxycarbonyl, non-aromatic heterocyclic alkyloxycarbonyl, aromatic carbocyclic alkylamino, non-aromatic Aromatic carbocyclic alkylamino, aromatic heterocyclic alkylamino, non-aromatic heterocyclic alkylamino, aromatic carbocyclic sulfanyl, non-aromatic carbocyclic sulfanyl, aromatic heterocyclic sulfanyl, non-aromatic heterocyclic sulfanyl, non-aromatic Aromatic carbocyclic sulfonyl, aromatic carbocyclic sulfonyl, aromatic heterocyclic sulfonyl, and non-aromatic heterocyclic sulfonyl.
　「置換若しくは非置換の芳香族炭素環式基」、「置換若しくは非置換の非芳香族炭素環式基」、「置換若しくは非置換の芳香族複素環式基」、「置換若しくは非置換の非芳香族複素環式基」、「置換若しくは非置換の芳香族炭素環アルキル」、「置換若しくは非置換の非芳香族炭素環アルキル」、「置換若しくは非置換の芳香族複素環アルキル」及び「置換若しくは非置換の非芳香族複素環アルキル」の「芳香族炭素環」、「非芳香族炭素環」、「芳香族複素環」及び「非芳香族複素環」の環上の置換基としては、次の置換基が挙げられる。環上の任意の位置の原子が次の置換基から選択される１以上の基と結合していてもよい。
　置換基：ハロゲン、ヒドロキシ、カルボキシ、アミノ、イミノ、ヒドロキシアミノ、ヒドロキシイミノ、ホルミル、ホルミルオキシ、カルバモイル、スルファモイル、スルファニル、スルフィノ、スルホ、チオホルミル、チオカルボキシ、ジチオカルボキシ、チオカルバモイル、シアノ、ニトロ、ニトロソ、アジド、ヒドラジノ、ウレイド、アミジノ、グアニジノ、トリアルキルシリル、アルキル、アルケニル、アルキニル、ハロアルキル、アルキルオキシ、アルケニルオキシ、アルキニルオキシ、ハロアルキルオキシ、アルキルオキシアルキル、アルキルオキシアルキルオキシ、アルキルカルボニル、アルケニルカルボニル、アルキニルカルボニル、モノアルキルアミノ、ジアルキルアミノ、アルキルスルホニル、アルケニルスルホニル、アルキニルスルホニル、モノアルキルカルボニルアミノ、ジアルキルカルボニルアミノ、モノアルキルスルホニルアミノ、ジアルキルスルホニルアミノ、アルキルイミノ、アルケニルイミノ、アルキニルイミノ、アルキルカルボニルイミノ、アルケニルカルボニルイミノ、アルキニルカルボニルイミノ、アルキルオキシイミノ、アルケニルオキシイミノ、アルキニルオキシイミノ、アルキルカルボニルオキシ、アルケニルカルボニルオキシ、アルキニルカルボニルオキシ、アルキルオキシカルボニル、アルケニルオキシカルボニル、アルキニルオキシカルボニル、アルキルスルファニル、アルケニルスルファニル、アルキニルスルファニル、アルキルスルフィニル、アルケニルスルフィニル、アルキニルスルフィニル、モノアルキルカルバモイル、ジアルキルカルバモイル、モノアルキルスルファモイル、ジアルキルスルファモイル、芳香族炭素環式基、非芳香族炭素環式基、芳香族複素環式基、非芳香族複素環式基、芳香族炭素環オキシ、非芳香族炭素環オキシ、芳香族複素環オキシ、非芳香族複素環オキシ、芳香族炭素環カルボニル、非芳香族炭素環カルボニル、芳香族複素環カルボニル、非芳香族複素環カルボニル、芳香族炭素環オキシカルボニル、非芳香族炭素環オキシカルボニル、芳香族複素環オキシカルボニル、非芳香族複素環オキシカルボニル、芳香族炭素環アルキル、非芳香族炭素環アルキル、芳香族複素環アルキル、非芳香族複素環アルキル、芳香族炭素環アルキルオキシ、非芳香族炭素環アルキルオキシ、芳香族複素環アルキルオキシ、非芳香族複素環アルキルオキシ、芳香族炭素環アルキルオキシカルボニル、非芳香族炭素環アルキルオキシカルボニル、芳香族複素環アルキルオキシカルボニル、非芳香族複素環アルキルオキシカルボニル、芳香族炭素環アルキルオキシアルキル、非芳香族炭素環アルキルオキシアルキル、芳香族複素環アルキルオキシアルキル、非芳香族複素環アルキルオキシアルキル、芳香族炭素環アルキルアミノ、非芳香族炭素環アルキルアミノ、芳香族複素環アルキルアミノ、非芳香族複素環アルキルアミノ、芳香族炭素環スルファニル、非芳香族炭素環スルファニル、芳香族複素環スルファニル、非芳香族複素環スルファニル、非芳香族炭素環スルホニル、芳香族炭素環スルホニル、芳香族複素環スルホニル、及び非芳香族複素環スルホニル。 “Substituted or unsubstituted aromatic carbocyclic group”, “substituted or unsubstituted non-aromatic carbocyclic group”, “substituted or unsubstituted aromatic heterocyclic group”, “substituted or unsubstituted non-substituted aromatic carbocyclic group” "Aromatic heterocyclic group", "Substituted or unsubstituted aromatic carbocyclic alkyl", "Substituted or unsubstituted non-aromatic carbocyclic alkyl", "Substituted or unsubstituted aromatic heterocyclic alkyl" and "Substituted Alternatively, the substituent on the ring of “aromatic carbocycle”, “non-aromatic carbocycle”, “aromatic heterocycle” and “non-aromatic heterocycle” of “unsubstituted non-aromatic heterocycle” is as follows: The following substituents are mentioned. An atom at any position on the ring may be bonded to one or more groups selected from the following substituents.
Substituents: halogen, hydroxy, carboxy, amino, imino, hydroxyamino, hydroxyimino, formyl, formyloxy, carbamoyl, sulfamoyl, sulfanyl, sulfino, sulfo, thioformyl, thiocarboxy, dithiocarboxy, thiocarbamoyl, cyano, nitro, nitroso , Azide, hydrazino, ureido, amidino, guanidino, trialkylsilyl, alkyl, alkenyl, alkynyl, haloalkyl, alkyloxy, alkenyloxy, alkynyloxy, haloalkyloxy, alkyloxyalkyl, alkyloxyalkyloxy, alkylcarbonyl, alkenylcarbonyl, Alkynylcarbonyl, monoalkylamino, dialkylamino, alkylsulfonyl, alkenylsulfonyl, Lucinylsulfonyl, monoalkylcarbonylamino, dialkylcarbonylamino, monoalkylsulfonylamino, dialkylsulfonylamino, alkylimino, alkenylimino, alkynylimino, alkylcarbonylimino, alkenylcarbonylimino, alkynylcarbonylimino, alkyloxyimino, alkenyloxyimino Alkynyloxyimino, alkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, alkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, alkylsulfanyl, alkenylsulfanyl, alkynylsulfanyl, alkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, monoalkylcarba Mo , Dialkylcarbamoyl, monoalkylsulfamoyl, dialkylsulfamoyl, aromatic carbocyclic group, non-aromatic carbocyclic group, aromatic heterocyclic group, non-aromatic heterocyclic group, aromatic carbocycle Oxy, non-aromatic carbocyclic oxy, aromatic heterocyclic oxy, non-aromatic heterocyclic oxy, aromatic carbocyclic carbonyl, non-aromatic carbocyclic carbonyl, aromatic heterocyclic carbonyl, non-aromatic heterocyclic carbonyl, aromatic Carbocyclic oxycarbonyl, non-aromatic carbocyclic oxycarbonyl, aromatic heterocyclic oxycarbonyl, non-aromatic heterocyclic oxycarbonyl, aromatic carbocyclic alkyl, non-aromatic carbocyclic alkyl, aromatic heterocyclic alkyl, non-aromatic Heterocyclic alkyl, aromatic carbocyclic alkyloxy, non-aromatic carbocyclic alkyloxy, aromatic heterocyclic alkyloxy, non-aromatic heterocyclic alkyl Oxy, aromatic carbocyclic alkyloxycarbonyl, non-aromatic carbocyclic alkyloxycarbonyl, aromatic heterocyclic alkyloxycarbonyl, non-aromatic heterocyclic alkyloxycarbonyl, aromatic carbocyclic alkyloxyalkyl, non-aromatic carbocyclic alkyl Oxyalkyl, aromatic heterocyclic alkyloxyalkyl, non-aromatic heterocyclic alkyloxyalkyl, aromatic carbocyclic alkylamino, non-aromatic carbocyclic alkylamino, aromatic heterocyclic alkylamino, non-aromatic heterocyclic alkylamino, Aromatic carbocyclic sulfanyl, non-aromatic carbocyclic sulfanyl, aromatic heterocyclic sulfanyl, non-aromatic heterocyclic sulfanyl, non-aromatic carbocyclic sulfonyl, aromatic carbocyclic sulfonyl, aromatic heterocyclic sulfonyl, and non-aromatic hetero Ring sulfonyl.
　「芳香族炭素環アルキル」、「非芳香族炭素環アルキル」、「芳香族複素環アルキル」、及び「非芳香族複素環アルキル」のアルキル部分も、上記「アルキル」と同様である。 The alkyl part of “aromatic carbocyclic alkyl”, “non-aromatic carbocyclic alkyl”, “aromatic heterocyclic alkyl”, and “non-aromatic heterocyclic alkyl” is the same as the above “alkyl”.
　「芳香族炭素環アルキル」とは、１以上の上記「芳香族炭素環式基」で置換されているアルキルを意味する。例えば、ベンジル、フェネチル、フェニルプロピニル、ベンズヒドリル、トリチル、ナフチルメチル、以下に示される基
Figure JPOXMLDOC01-appb-C000003
等が挙げられる。
　「芳香族炭素環アルキル」の好ましい態様としては、ベンジル、フェネチル、ベンズヒドリルが挙げられる。 “Aromatic carbocyclic alkyl” means an alkyl substituted with one or more of the above “aromatic carbocyclic groups”. For example, benzyl, phenethyl, phenylpropynyl, benzhydryl, trityl, naphthylmethyl, groups shown below
Figure JPOXMLDOC01-appb-C000003
Etc.
Preferable embodiments of “aromatic carbocyclic alkyl” include benzyl, phenethyl and benzhydryl.
　「非芳香族炭素環アルキル」とは、１以上の上記「非芳香族炭素環式基」で置換されているアルキルを意味する。また、「非芳香族炭素環アルキル」は、アルキル部分が上記「芳香族炭素環式基」で置換されている「非芳香族炭素環アルキル」も包含する。例えば、シクロプロピルメチル、シクロブチルメチル、シクロペンチルメチル、シクロへキシルメチル、以下に示される基
Figure JPOXMLDOC01-appb-C000004
等が挙げられる。 “Non-aromatic carbocyclic alkyl” means alkyl substituted with one or more of the above “non-aromatic carbocyclic groups”. The “non-aromatic carbocyclic alkyl” also includes “non-aromatic carbocyclic alkyl” in which the alkyl moiety is substituted with the above “aromatic carbocyclic group”. For example, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, groups shown below
Figure JPOXMLDOC01-appb-C000004
Etc.
　「芳香族複素環アルキル」とは、１以上の上記「芳香族複素環式基」で置換されているアルキルを意味する。また、「芳香族複素環アルキル」は、アルキル部分が上記「芳香族炭素環式基」及び／又は「非芳香族炭素環式基」で置換されている「芳香族複素環アルキル」も包含する。例えば、ピリジルメチル、フラニルメチル、イミダゾリルメチル、インドリルメチル、ベンゾチオフェニルメチル、オキサゾリルメチル、イソキサゾリルメチル、チアゾリルメチル、イソチアゾリルメチル、ピラゾリルメチル、イソピラゾリルメチル、ピロリジニルメチル、ベンズオキサゾリルメチル、以下に示される基
Figure JPOXMLDOC01-appb-C000005
等が挙げられる。 “Aromatic heterocyclic alkyl” means alkyl substituted with one or more of the above “aromatic heterocyclic groups”. “Aromatic heterocyclic alkyl” also includes “aromatic heterocyclic alkyl” in which the alkyl moiety is substituted with the above “aromatic carbocyclic group” and / or “non-aromatic carbocyclic group”. . For example, pyridylmethyl, furanylmethyl, imidazolylmethyl, indolylmethyl, benzothiophenylmethyl, oxazolylmethyl, isoxazolylmethyl, thiazolylmethyl, isothiazolylmethyl, pyrazolylmethyl, isopyrazolylmethyl, pyrrolidinylmethyl, benz Oxazolylmethyl, group shown below
Figure JPOXMLDOC01-appb-C000005
Etc.
　「非芳香族複素環アルキル」とは、１以上の上記「非芳香族複素環式基」で置換されているアルキルを意味する。また、「非芳香族複素環アルキル」は、アルキル部分が上記「芳香族炭素環式基」、「非芳香族炭素環式基」及び／又は「芳香族複素環式基」で置換されている「非芳香族複素環アルキル」も包含する。例えば、テトラヒドロピラニルメチル、モルホリニルエチル、ピペリジニルメチル、ピペラジニルメチル、以下に示される基
Figure JPOXMLDOC01-appb-C000006
等が挙げられる。 “Non-aromatic heterocyclic alkyl” means an alkyl substituted with one or more of the above “non-aromatic heterocyclic groups”. In the “non-aromatic heterocyclic alkyl”, the alkyl portion is substituted with the above “aromatic carbocyclic group”, “non-aromatic carbocyclic group” and / or “aromatic heterocyclic group”. Also included are “non-aromatic heterocyclic alkyl”. For example, tetrahydropyranylmethyl, morpholinylethyl, piperidinylmethyl, piperazinylmethyl, groups shown below
Figure JPOXMLDOC01-appb-C000006
Etc.
　本発明のアンチセンスオリゴヌクレオチド（又はその修飾体）は常法によって合成することができ、例えば、市販の核酸自動合成装置（例えば、ＡｐｐｌｉｅｄＢｉｏｓｙｓｔｅｍｓ社製、（株）大日本精機製等）によって容易に合成することができる。合成法はホスホロアミダイトを用いた固相合成法、ハイドロジェンホスホネートを用いた固相合成法等がある。例えば、Ｔｅｔｒａｈｅｄｒｏｎ　Ｌｅｔｔｅｒｓ　２２，　１８５９－１８６２　（１９８１）、国際公開第２０１１／０５２４３６号等に開示されている。 The antisense oligonucleotide of the present invention (or a modified product thereof) can be synthesized by a conventional method. For example, it can be easily synthesized with a commercially available automatic nucleic acid synthesizer (for example, manufactured by AppliedBiosystems, manufactured by Dainippon Seiki Co., Ltd.). Can be synthesized. Examples of the synthesis method include a solid phase synthesis method using phosphoramidite and a solid phase synthesis method using hydrogen phosphonate. For example, it is disclosed in Tetrahedron Letters 22, 1859-1862 (1981), International Publication No. 2011/052436, and the like.
　本発明のアンチセンスオリゴヌクレオチドは、ヒトを含む動物に投与する際、生物学的に活性な代謝産物又はその残渣物を（直接的に、あるいは、間接的に）提供し得る、任意の製薬上許容される塩、エステル、若しくはかかるエステルの塩、又は任意の他の同等物を包含する。つまり、本発明のアンチセンスオリゴヌクレオチドのプロドラッグ及び製薬上許容される塩、該プロドラッグの製薬上許容される塩、ならびに他の生物学的同等物を包含する。 The antisense oligonucleotides of the present invention can be any pharmaceutical that can provide (directly or indirectly) a biologically active metabolite or residue thereof when administered to an animal, including a human. Acceptable salts, esters, or salts of such esters, or any other equivalent. That is, it includes prodrugs and pharmaceutically acceptable salts of the antisense oligonucleotides of the invention, pharmaceutically acceptable salts of the prodrugs, and other biological equivalents.
　「プロドラッグ」とは、内在性酵素又は他の化学物質の作用及び／又は状態によってその生体内又は細胞内で活性形態（即ち、薬物）に変換される、不活性形態又はより低い活性形態の誘導体である。本発明のアンチセンスオリゴヌクレオチドのプロドラッグは、国際公開第９３／２４５１０号、国際公開第９４／２６７６４号等に記載される方法に従って調製することができる。 A “prodrug” is an inactive or less active form that is converted into an active form (ie, drug) in the body or cell by the action and / or state of an endogenous enzyme or other chemical. Is a derivative. The prodrug of the antisense oligonucleotide of the present invention can be prepared according to the methods described in WO 93/24510, WO 94/26764 and the like.
　「製薬上許容される塩」とは、本発明のアンチセンスオリゴヌクレオチドの生理学的に及び製薬上許容される塩、即ち、該アンチセンスオリゴヌクレオチドの所望される生物学的な活性を保持し、そこで望まれない毒物学的効果を与えない塩のことをいう。 “Pharmaceutically acceptable salt” refers to a physiologically and pharmaceutically acceptable salt of an antisense oligonucleotide of the invention, ie, retains the desired biological activity of the antisense oligonucleotide; It refers to salts that do not give unwanted toxicological effects.
　製薬上許容される塩としては、例えば、アルカリ金属（例えば、リチウム、ナトリウム、カリウム等）、アルカリ土類金属（例えば、カルシウム、バリウム等）、マグネシウム、遷移金属（例えば、亜鉛、鉄等）、アンモニア、有機塩基（例えば、トリメチルアミン、トリエチルアミン、ジシクロヘキシルアミン、エタノールアミン、ジエタノールアミン、トリエタノールアミン、メグルミン、ジエタノールアミン、エチレンジアミン、ピリジン、ピコリン、キノリン等）及びアミノ酸との塩、又は無機酸（例えば、塩酸、硫酸、硝酸、炭酸、臭化水素酸、リン酸、ヨウ化水素酸等）、及び有機酸（例えば、ギ酸、酢酸、プロピオン酸、トリフルオロ酢酸、クエン酸、乳酸、酒石酸、シュウ酸、マレイン酸、フマル酸、マンデル酸、グルタル酸、リンゴ酸、安息香酸、フタル酸、アスコルビン酸、ベンゼンスルホン酸、ｐ－トルエンスルホン酸、メタンスルホン酸、エタンスルホン酸等）との塩が挙げられる。特に塩酸、硫酸、リン酸、酒石酸、メタンスルホン酸との塩等が挙げられる。これらの塩は、通常行われる方法によって形成させることができる。 Examples of pharmaceutically acceptable salts include alkali metals (eg, lithium, sodium, potassium, etc.), alkaline earth metals (eg, calcium, barium, etc.), magnesium, transition metals (eg, zinc, iron, etc.), Ammonia, organic bases (eg trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, meglumine, diethanolamine, ethylenediamine, pyridine, picoline, quinoline etc.) and salts with amino acids, or inorganic acids (eg hydrochloric acid, Sulfuric acid, nitric acid, carbonic acid, hydrobromic acid, phosphoric acid, hydroiodic acid, etc.) and organic acids (eg formic acid, acetic acid, propionic acid, trifluoroacetic acid, citric acid, lactic acid, tartaric acid, oxalic acid, maleic acid) , Fumaric acid, mandelic acid, gluta Acid, malic acid, benzoic acid, phthalic acid, ascorbic acid, benzenesulfonic acid, p- toluenesulfonic acid, methanesulfonic acid, and salts with ethanesulfonic acid, etc.). Particularly, salts with hydrochloric acid, sulfuric acid, phosphoric acid, tartaric acid, methanesulfonic acid and the like can be mentioned. These salts can be formed by a commonly performed method.
　本発明は、本発明のアンチセンスオリゴヌクレオチドを含有する医薬組成物も包含する。本発明の医薬組成物の投与方法及び製剤は、当該分野で公知の投与方法及び製剤であれば、いずれも利用可能である。アンチセンスオリゴヌクレオチドの投与方法及び製剤は、例えば、以下の文献にも開示されている。
国際公開第２００４／０１６７４９号、国際公開第２００５／０８３１２４号、国際公開２００７／１４３３１５号、国際公開第２００９／０７１６８０号、国際公開第２０１３／０８９２８３号等。 The present invention also includes a pharmaceutical composition containing the antisense oligonucleotide of the present invention. As the administration method and preparation of the pharmaceutical composition of the present invention, any administration method and preparation known in the art can be used. Antisense oligonucleotide administration methods and preparations are also disclosed in, for example, the following documents.
International Publication No. 2004/016749, International Publication No. 2005/083124, International Publication No. 2007/143315, International Publication No. 2009/071680, International Publication No. 2013/0889283, and the like.
　本発明のアンチセンスオリゴヌクレオチドを含有する医薬組成物としては、例えば、本発明のアンチセンスオリゴヌクレオチドに相補的な核酸とを含む二重鎖核酸が挙げられる。該二重鎖核酸は、アンチセンスオリゴヌクレオチドを特異性高く効率的に標的部位に送達し、かつ該アンチセンスオリゴヌクレオチドにより、標的遺伝子の発現を効果的に抑制することを可能とする。該二重鎖核酸の具体例及びその調製方法は、国際公開第２０１３／０８９２８３号に記載されている。 Examples of the pharmaceutical composition containing the antisense oligonucleotide of the present invention include a double-stranded nucleic acid containing a nucleic acid complementary to the antisense oligonucleotide of the present invention. The double-stranded nucleic acid enables an antisense oligonucleotide to be delivered to a target site with high specificity and efficiently, and enables the expression of a target gene to be effectively suppressed by the antisense oligonucleotide. Specific examples of the double-stranded nucleic acid and a method for preparing the double-stranded nucleic acid are described in International Publication No. 2013/0889283.
　本発明の医薬組成物は、局所的あるいは全身的な治療のいずれが望まれるのか、又は治療すべき領域に応じて、様々な方法により投与することができる。投与方法としては、例えば、局所的（点眼、膣内、直腸内、鼻腔内、経皮を含む）、経口的、又は、非経口的であってもよい。非経口的投与としては、静脈内注射若しくは点滴、皮下、腹腔内若しくは筋肉内注入、吸引若しくは吸入による肺投与、硬膜下腔内投与、脳室内投与等が挙げられる。 The pharmaceutical composition of the present invention can be administered by various methods depending on whether local or systemic treatment is desired or on the region to be treated. The administration method may be, for example, topical (including eye drops, intravaginal, rectal, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous injection or infusion, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration by inhalation or inhalation, intradural administration, intraventricular administration, and the like.
　本発明の医薬組成物を局所投与する場合、経皮パッチ、軟膏、ローション、クリーム、ゲル、滴下剤、坐剤、噴霧剤、液剤、散剤等の製剤を用いることができる。
　経口投与用組成物としては、散剤、顆粒剤、水若しくは非水性媒体に溶解させた懸濁液又は溶液、カプセル、粉末剤、錠剤等が挙げられる。
　非経口、硬膜下腔、又は、脳室内投与用組成物としては、バッファー、希釈剤及びその他の適当な添加剤を含む無菌水溶液等が挙げられる。 When the pharmaceutical composition of the present invention is administered locally, preparations such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders and the like can be used.
Examples of compositions for oral administration include powders, granules, suspensions or solutions dissolved in water or non-aqueous media, capsules, powders, tablets and the like.
Examples of compositions for parenteral, subdural space, or intraventricular administration include sterile aqueous solutions containing buffers, diluents and other suitable additives.
　本発明の医薬組成物は、本発明のアンチセンスオリゴヌクレオチドの有効量にその剤型に適した賦形剤、結合剤、湿潤剤、崩壊剤、滑沢剤、希釈剤等の各種医薬用添加剤を必要に応じて混合して得ることができる。注射剤の場合には適当な担体と共に滅菌処理を行なって製剤とすればよい。 The pharmaceutical composition of the present invention comprises various pharmaceutical additives such as excipients, binders, wetting agents, disintegrants, lubricants, diluents and the like suitable for the dosage form in the effective amount of the antisense oligonucleotide of the present invention. The agent can be obtained by mixing as necessary. In the case of an injection, it may be sterilized with an appropriate carrier to form a preparation.
　賦形剤としては乳糖、白糖、ブドウ糖、デンプン、炭酸カルシウム又は結晶セルロース等が挙げられる。結合剤としてはメチルセルロース、カルボキシメチルセルロース、ヒドロキシプロピルセルロース、ゼラチン又はポリビニルピロリドン等が挙げられる。崩壊剤としてはカルボキシメチルセルロース、カルボキシメチルセルロースナトリウム、デンプン、アルギン酸ナトリウム、カンテン末又はラウリル硫酸ナトリウム等が挙げられる。滑沢剤としてはタルク、ステアリン酸マグネシウム又はマクロゴール等が挙げられる。坐剤の基剤としてはカカオ脂、マクロゴール又はメチルセルロース等を用いることができる。また、液剤又は乳濁性、懸濁性の注射剤として調製する場合には通常使用されている溶解補助剤、懸濁化剤、乳化剤、安定化剤、保存剤、等張剤等を適宜添加しても良い。経口投与の場合には嬌味剤、芳香剤等を加えても良い。 Excipients include lactose, sucrose, glucose, starch, calcium carbonate or crystalline cellulose. Examples of the binder include methyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, gelatin, and polyvinyl pyrrolidone. Examples of the disintegrant include carboxymethyl cellulose, sodium carboxymethyl cellulose, starch, sodium alginate, agar powder, or sodium lauryl sulfate. Examples of the lubricant include talc, magnesium stearate or macrogol. As a suppository base, cocoa butter, macrogol, methylcellulose, or the like can be used. In addition, when preparing as liquid or emulsion or suspension injections, commonly used solubilizers, suspending agents, emulsifiers, stabilizers, preservatives, isotonic agents, etc. are added as appropriate. You may do it. In the case of oral administration, flavoring agents, fragrances and the like may be added.
　投与は、治療される病態の重度と反応度に依存し、治療コースは、数日から数ヶ月、あるいは、治癒が実現されるまで、又は、病状の減退が達成されるまで持続する。最適投与スケジュールは、生体における薬剤蓄積の測定から計算が可能である。当該分野の当業者であれば、最適用量、投与法、及び、繰り返し頻度を定めることができる。最適用量は、個々のアンチセンスオリゴヌクレオチドの相対的効力に応じて変動するが、一般に、インビトロ及びインビボの動物実験におけるＩＣ５０又はＥＣ５０に基づいて計算することが可能である。例えば、アンチセンスオリゴヌクレオチドの分子量（アンチセンスオリゴヌクレオチド配列及び化学構造から導かれる）と、例えば、ＩＣ５０のような効果的用量（実験的に導かれる）が与えられたならば、ｍｇ／ｋｇで表される用量が通例に従って計算される。 Administration depends on the severity and responsiveness of the condition being treated, and the course of treatment lasts from days to months, or until healing is achieved, or until disease status is achieved. The optimal dosing schedule can be calculated from measurements of drug accumulation in the body. Persons of ordinary skill in the art can determine optimum dosages, dosing methodologies and repetition rates. The optimal dose will vary depending on the relative potency of the individual antisense oligonucleotides, but can generally be calculated based on the IC50 or EC50 in in vitro and in vivo animal experiments. For example, given the molecular weight of an antisense oligonucleotide (derived from the antisense oligonucleotide sequence and chemical structure) and an effective dose (derived experimentally) such as IC50, for example, mg / kg The expressed dose is customarily calculated.
　本発明の医薬組成物は、ＡＣＳＬ１発現抑制活性を有するため、ＡＣＳＬ１が関連する疾患の予防又は治療のために用いることができる。
　ＡＣＳＬ１が関連する疾患としては、肥満症（肥満症における体重管理も含む）、肥満関連疾患、糖尿病（特に、ＩＩ型糖尿病）、シンドロームＸ、心臓血管障害又は癌（乳癌、結腸癌、大腸癌、卵巣癌、肺癌等）が挙げられる。
　「肥満関連疾患」とは、肥満に伴うか、肥満により引き起こされるか、肥満の結果起きる疾患である。肥満関連疾患の例としては、過食症、高血圧、耐糖能異常、糖尿病、代謝症候群、脂質代謝異常、動脈硬化症、高尿酸血症、痛風、脂肪肝、蛋白尿、肥満腎症、子宮内膜癌、乳癌、前立腺癌、大腸癌、変形性関節症、腰痛症、腰椎症、閉塞性睡眠時無呼吸症候群、冠動脈疾患（心筋梗塞、狭心症等の冠動脈性心疾患）、脳梗塞、脳血栓症、一過的脳虚血発作、月経異常、プラダーウィリー症候群、フレーリッヒ症候群、ピックウィック症候群等が挙げられる。本発明の医薬組成物は左心室肥大のリスクの低減等、肥満の２次的な結果のリスクの低減にも有用である。
　本発明の医薬組成物は、特に、肥満症又はＩＩ型糖尿病の予防又は治療のために用いることができる。 Since the pharmaceutical composition of the present invention has ACSL1 expression-suppressing activity, it can be used for prevention or treatment of diseases associated with ACSL1.
ACSL1 related diseases include obesity (including weight management in obesity), obesity related diseases, diabetes (especially type II diabetes), syndrome X, cardiovascular disorder or cancer (breast cancer, colon cancer, colon cancer, Ovarian cancer, lung cancer, etc.).
An “obesity related disease” is a disease associated with, caused by, or caused by obesity. Examples of obesity-related diseases include bulimia, hypertension, impaired glucose tolerance, diabetes, metabolic syndrome, lipid metabolism disorder, arteriosclerosis, hyperuricemia, gout, fatty liver, proteinuria, obese nephropathy, endometrium Cancer, breast cancer, prostate cancer, colon cancer, osteoarthritis, low back pain, lumbar spondylosis, obstructive sleep apnea syndrome, coronary artery disease (myocardial infarction, coronary heart disease such as angina pectoris), cerebral infarction, cerebral thrombus Disease, transient cerebral ischemic attack, menstrual abnormalities, Prader-Willi syndrome, Frehrich syndrome, Pickwick syndrome and the like. The pharmaceutical composition of the present invention is also useful for reducing the risk of secondary outcomes of obesity, such as reducing the risk of left ventricular hypertrophy.
The pharmaceutical composition of the present invention can be used particularly for the prevention or treatment of obesity or type II diabetes.
　本発明の医薬組成物を肥満症の予防又は治療のために用いる場合、他の１若しくは複数の公知の抗肥満薬（抗肥満作用を有する化合物を含有する医薬組成物、肥満症や肥満症における体重管理等に用いることのできる薬剤）と組み合わせて用いることもできる。また、本発明の医薬組成物の投与療法は、既知の食事療法、薬物療法、運動等と組み合わせて用いることもできる。 When the pharmaceutical composition of the present invention is used for the prevention or treatment of obesity, other one or more known anti-obesity drugs (pharmaceutical compositions containing a compound having an anti-obesity action, obesity and obesity) It can also be used in combination with a drug that can be used for weight management and the like. Moreover, the administration therapy of the pharmaceutical composition of the present invention can also be used in combination with known diet therapy, drug therapy, exercise and the like.
　例えば、以下の方法も本発明の範囲内である。
　本発明の医薬組成物と併用して、公知の抗肥満薬を投与することを特徴とする、肥満若しくは肥満関連疾患の予防若しくは治療又は肥満における体重管理の方法。
　本発明の医薬組成物の投与による予防又は治療を受けている患者に、公知の抗肥満薬を投与することを特徴とする、肥満若しくは肥満関連疾患の予防若しくは治療又は肥満における体重管理の方法。 For example, the following methods are also within the scope of the present invention.
A method for the prevention or treatment of obesity or obesity-related diseases or weight management in obesity, comprising administering a known anti-obesity drug in combination with the pharmaceutical composition of the present invention.
A method for the prevention or treatment of obesity or obesity-related diseases or weight management in obesity, comprising administering a known anti-obesity drug to a patient undergoing prevention or treatment by administration of the pharmaceutical composition of the present invention.
　公知の抗肥満薬としては、食欲抑制作用を有する化合物（選択的セロトニン再取り込み阻害剤等）、栄養素の消化吸収抑制作用を有する化合物（α‐グルコシダーゼ阻害剤；ＳＧＬＴ－２阻害剤等）、脂肪吸収抑制作用を有する化合物（リパーゼ阻害剤；胆汁酸吸着レジン等）、５ＨＴトランスポーター阻害剤、ＮＥトランスポーター阻害剤、ＣＢ－１アンタゴニスト／インバースアゴニスト、グレリンアンタゴニスト、Ｈ３アンタゴニスト／インバースアゴニスト、ＭＣＨ　Ｒ１アンタゴニスト、ＭＣＨ　Ｒ２アゴニスト／アンタゴニスト、ＮＰＹ　Ｙ１受容体 アンタゴニスト、ＮＰＹ　Ｙ２受容体アゴニスト、ＮＰＹ　Ｙ４受容体アゴニスト、ＮＰＹ　Ｙ５受容体アンタゴニスト、ｍＧｌｕＲ５アンタゴニスト、レプチン、レプチンアゴニスト、レプチン誘導体、オピオイドアンタゴニスト、オレキシンアンタゴニスト、ＢＲＳ３アゴニスト、ＣＣＫ－Ａアゴニスト、ＣＮＴＦ、ＣＮＴＦアゴニスト、ＣＮＴＦ誘導体、ＧＨＳアゴニスト、５ＨＴ２Ｃアゴニスト、Ｍｃ４ｒアゴニスト、モノアミン再取り込み阻害剤、ＧＬＰ－１アゴニスト、ＵＣＰ－１、２及び３活性剤、β３アゴニスト、甲状腺ホルモンβアゴニスト、ＰＤＥ阻害剤、ＦＡＳ阻害剤、ＤＧＡＴ１阻害剤、ＤＧＡＴ２阻害剤、ＡＣＣ２阻害剤、グルココルチコイドアンタゴニスト、アシル－エストロゲン、脂肪酸トランスポーター阻害剤、ジカルボン酸トランスポーター阻害剤等を含有する医薬組成物が挙げられる。 Known anti-obesity drugs include compounds having an appetite suppressing action (selective serotonin reuptake inhibitors, etc.), compounds having an action to suppress digestion and absorption of nutrients (α-glucosidase inhibitors; SGLT-2 inhibitors, etc.), fat Compounds having absorption-inhibiting action (lipase inhibitor; bile acid adsorption resin, etc.), 5HT transporter inhibitor, NE transporter inhibitor, CB-1 antagonist / inverse agonist, ghrelin antagonist, H3 antagonist / inverse agonist, MCH R1 antagonist , MCH R2 agonist / antagonist, NPY Y1 receptor antagonist, NPY Y2 receptor agonist, NPY Y4 receptor agonist, NPY Y5 receptor antagonist, mGluR5 antagonist, leptin, leptin Gonist, leptin derivative, opioid antagonist, orexin antagonist, BRS3 agonist, CCK-A agonist, CNTF, CNTF agonist, CNTF derivative, GHS agonist, 5HT2C agonist, Mc4r agonist, monoamine reuptake inhibitor, GLP-1 agonist, UCP-1 2 and 3 activators, β3 agonists, thyroid hormone β agonists, PDE inhibitors, FAS inhibitors, DGAT1 inhibitors, DGAT2 inhibitors, ACC2 inhibitors, glucocorticoid antagonists, acyl-estrogens, fatty acid transporter inhibitors, dicarboxylic Examples include pharmaceutical compositions containing an acid transporter inhibitor and the like.
　本発明の医薬組成物をＩＩ型糖尿病の予防又は治療のために用いる場合、他の１若しくは複数の公知のＩＩ型糖尿病治療薬と組み合わせて用いることもできる。 When the pharmaceutical composition of the present invention is used for the prevention or treatment of type II diabetes, it can also be used in combination with one or more other known type II diabetes therapeutic agents.
　公知のＩＩ型糖尿病治療薬としては、インスリン分泌促進薬（例えば、スルホニル尿素（ＳＵ）薬）、速効型インスリン分泌促進薬（例えば、フェニルアラニン誘導体薬）、ブドウ糖吸収阻害薬（例えば、αグルコシダーゼ阻害薬（αＧＩ薬））、インスリン抵抗性改善薬（例えば、ビグアナイド系薬剤（ＢＧ薬）、チアゾリジン系誘導体（ＴＺＤ薬））、インスリン製剤、ペプチジルペプチダーゼＩＶ（ＤＰＰ－ＩＶ）阻害薬、ＧＬＰ－１受容体アゴニスト、１型ナトリウム依存性グルコース輸送体（ＳＧＬＴ１）阻害薬、２型ナトリウム依存性グルコース輸送体（ＳＧＬＴ２）阻害薬等を含有する医薬組成物が挙げられる。 Known therapeutic agents for type II diabetes include insulin secretagogues (for example, sulfonylurea (SU) drugs), fast-acting insulin secretagogues (for example, phenylalanine derivative drugs), glucose absorption inhibitors (for example, α-glucosidase inhibitors) (ΑGI drug)), insulin sensitizers (for example, biguanide drugs (BG drugs), thiazolidine derivatives (TZD drugs)), insulin preparations, peptidyl peptidase IV (DPP-IV) inhibitors, GLP-1 receptors Examples include pharmaceutical compositions containing agonists, type 1 sodium-dependent glucose transporter (SGLT1) inhibitors, type 2 sodium-dependent glucose transporter (SGLT2) inhibitors, and the like.
　本発明の医薬組成物と他の薬剤とを組み合わせて用いる場合、投与時期は限定されず、投与対象に対し、同時に投与してもよいし、時間差をおいて投与してもよい。さらに、本発明の医薬組成物と該他の薬剤とは、それぞれの活性成分を含む複数の製剤として投与されてもよいし、両方の活性成分を含む単一の製剤として投与されてもよい。 When the pharmaceutical composition of the present invention is used in combination with another drug, the timing of administration is not limited, and it may be administered simultaneously to the administration subject or may be administered with a time difference. Furthermore, the pharmaceutical composition of the present invention and the other drug may be administered as a plurality of preparations containing each active ingredient, or may be administered as a single preparation containing both active ingredients.
　以下に本発明の実施例を挙げて本発明をさらに詳しく説明するが、本発明はこれらにより限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples of the present invention, but the present invention is not limited thereto.
実施例１：オリゴヌクレオチド合成
　本発明に関連するオリゴヌクレオチドは、Ｔｅｔｒａｈｅｄｒｏｎ　Ｌｅｔｔｅｒｓ　２２，１８５９‐１８６２　（１９８１）、国際公開第２０１１／０５２４３６号等に記載される方法によって合成した。 Example 1 Oligonucleotide Synthesis Oligonucleotides related to the present invention were synthesized by the method described in Tetrahedron Letters 22, 1859-1862 (1981), International Publication No. 2011/052436, and the like.
　具体的には、式（ａ）で示されるＬＮＡ又は式（ｂ）で示される２’－ＯＭｅを含むオリゴヌクレオチドに関しては、株式会社ジーンデザインに合成委託した。
Figure JPOXMLDOC01-appb-C000007
（式中、Ｂａｓｅは５－メチルシトシン（Ｃ）、チミン（Ｔ）、アデニン（Ａ）又はグアニン（Ｇ）である。）
Figure JPOXMLDOC01-appb-C000008
（式中、Ｂａｓｅは、シトシン（Ｃ）、ウラシル（Ｕ）、アデニン（Ａ）又はグアニン（Ｇ）であり、Ｍｅはメチルである。） Specifically, synthesis of LNA represented by the formula (a) or 2′-OMe represented by the formula (b) was outsourced to Gene Design Co., Ltd.
Figure JPOXMLDOC01-appb-C000007
(In the formula, Base is 5-methylcytosine (C), thymine (T), adenine (A) or guanine (G).)
Figure JPOXMLDOC01-appb-C000008
(In the formula, Base is cytosine (C), uracil (U), adenine (A) or guanine (G), and Me is methyl.)
　式（ｃ）で示されるアミドＢＮＡ（ＡｍＮＡ）を含むオリゴヌクレオチドに関しては、国際公開第２０１１／０５２４３６号に記載の方法を参照して合成した。
Figure JPOXMLDOC01-appb-C000009
（式中、Ｂａｓｅは、５－メチルシトシン（Ｃ）、チミン（Ｔ）、アデニン（Ａ）又はグアニン（Ｇ）であり、Ｍｅはメチルである。） The oligonucleotide containing amide BNA (AmNA) represented by the formula (c) was synthesized with reference to the method described in WO 2011/052436.
Figure JPOXMLDOC01-appb-C000009
(In the formula, Base is 5-methylcytosine (C), thymine (T), adenine (A) or guanine (G), and Me is methyl.)
　式（ａ）で示されるＬＮＡ、式（ｂ）で示される２’－ＯＭｅ又は式（ｃ）で示されるアミドＢＮＡ（ＡｍＮＡ）を含有する１０ｍｅｒ～１９ｍｅｒのオリゴヌクレオチドは、核酸自動合成機（ｎＳ－８型、（株）大日本精機製）を用いて、０．２μｍｏｌスケールで合成した。鎖長の伸長は標準的なホスホロアミダイトプロトコール（固相担体：ＣＰＧレジン、硫化はＤＤＴ（３Ｈ－１，２－Ｂｅｎｚｏｄｉｔｈｉｏｌｅ－３－ｏｎｅ，　１，１－ｄｉｏｘｉｄｅ）等を使用）にて実施し、末端の５’位の水酸基がＤＭＴｒ（ジメトキシトリチル）基で保護され、かつ３’位が固相に担持されたオリゴヌクレオチドを得た。続いて、酸処理により、ＤＭＴｒ基を除去した後、塩基処理することにより、目的物を固相担体から切り出した。希酸にて中和後、溶媒を留去し、得られた粗生成物をゲルろ過カラムクロマト、逆相ＨＰＬＣにて精製することにより目的物を得た。 A 10mer to 19mer oligonucleotide containing an LNA represented by the formula (a), 2′-OMe represented by the formula (b) or an amide BNA (AmNA) represented by the formula (c) is used in an automatic nucleic acid synthesizer (nS -8 type, manufactured by Dainippon Seiki Co., Ltd.) and was synthesized on a 0.2 μmol scale. Chain length extension is carried out using a standard phosphoramidite protocol (solid phase carrier: CPG resin, sulfurization using DDT (3H-1,2-Benzodithiole-3-one, 1,1-dioxide), etc.) Thus, an oligonucleotide in which the terminal 5′-position hydroxyl group was protected with a DMTr (dimethoxytrityl) group and the 3′-position was supported on a solid phase was obtained. Subsequently, the DMTr group was removed by acid treatment, followed by base treatment to cut out the target product from the solid phase carrier. After neutralization with dilute acid, the solvent was distilled off, and the resulting crude product was purified by gel filtration column chromatography and reverse phase HPLC to obtain the desired product.
実施例２：ヒト又はマウスアンチセンスオリゴヌクレオチド配列
　アンチセンスオリゴヌクレオチド（ＡＯＮ）を、ヒトＡＣＳＬ１（ＧｅｎＢａｎｋ：ＮＭ_００１９９５、配列番号１）又はマウスＡｃｓｌ１（ＧｅｎＢａｎｋ：ＮＭ_００７９８１、配列番号：３）を標的とするよう設計した。オリゴヌクレオチド配列を表１～２６に示す。表の標的部位のカラム中のｍは、マウスの配列の標的部位を示し、ｈはヒトの配列の標的部位を示す。
　表１～２０の配列中、大文字は式（ａ）で示されるＬＮＡを表す。小文字はＤＮＡを表す。ヌクレオシド間の結合は、オリゴヌクレオチド全体を通してホスホロチオエート（Ｐ＝Ｓ）である。 Example 2: Human or mouse antisense oligonucleotide sequence Antisense oligonucleotide (AON) is targeted to human ACSL1 (GenBank: NM_001995, SEQ ID NO: 1) or mouse Acsl1 (GenBank: NM_007981, SEQ ID NO: 3) Designed. The oligonucleotide sequences are shown in Tables 1-26. In the target site column in the table, m represents the target site of the mouse sequence, and h represents the target site of the human sequence.
In the sequences of Tables 1 to 20, capital letters represent LNAs represented by formula (a). Lower case letters represent DNA. The linkage between nucleosides is phosphorothioate (P = S) throughout the oligonucleotide.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000014
Figure JPOXMLDOC01-appb-T000014
Figure JPOXMLDOC01-appb-T000015
Figure JPOXMLDOC01-appb-T000015
Figure JPOXMLDOC01-appb-T000016
Figure JPOXMLDOC01-appb-T000016
Figure JPOXMLDOC01-appb-T000017
Figure JPOXMLDOC01-appb-T000017
Figure JPOXMLDOC01-appb-T000018
Figure JPOXMLDOC01-appb-T000018
Figure JPOXMLDOC01-appb-T000019
Figure JPOXMLDOC01-appb-T000019
Figure JPOXMLDOC01-appb-T000020
Figure JPOXMLDOC01-appb-T000020
Figure JPOXMLDOC01-appb-T000021
Figure JPOXMLDOC01-appb-T000021
Figure JPOXMLDOC01-appb-T000022
Figure JPOXMLDOC01-appb-T000022
Figure JPOXMLDOC01-appb-T000023
Figure JPOXMLDOC01-appb-T000023
Figure JPOXMLDOC01-appb-T000024
Figure JPOXMLDOC01-appb-T000024
Figure JPOXMLDOC01-appb-T000025
Figure JPOXMLDOC01-appb-T000025
Figure JPOXMLDOC01-appb-T000026
Figure JPOXMLDOC01-appb-T000026
Figure JPOXMLDOC01-appb-T000027
Figure JPOXMLDOC01-appb-T000027
Figure JPOXMLDOC01-appb-T000028
Figure JPOXMLDOC01-appb-T000028
Figure JPOXMLDOC01-appb-T000029
Figure JPOXMLDOC01-appb-T000029
　表２１の配列中、大文字は式（ａ）で示されるＬＮＡを表す。小文字はＤＮＡを表す。ヌクレオシド間の結合は、オリゴヌクレオチド全体を通してホスホロチオエート（Ｐ＝Ｓ）である。また、Ａｅ、Ｇｅは、式（ｂ）で示される２’－ＯＭｅを表す。
Figure JPOXMLDOC01-appb-T000030
 In the sequences of Table 21, uppercase letters represent LNAs represented by formula (a). Lower case letters represent DNA. The linkage between nucleosides is phosphorothioate (P = S) throughout the oligonucleotide. A e and G e represent 2′-OMe represented by the formula (b).
Figure JPOXMLDOC01-appb-T000030
　表２２～２３の配列中、大文字は式（ｃ）で示されるアミドＢＮＡ（ＡｍＮＡ）を表す。小文字はＤＮＡを表す。ヌクレオシド間の結合は、オリゴヌクレオチド全体を通してホスホロチオエート（Ｐ＝Ｓ）である。


In the sequences of Tables 22 to 23, capital letters represent amide BNA (AmNA) represented by the formula (c). Lower case letters represent DNA. The linkage between nucleosides is phosphorothioate (P = S) throughout the oligonucleotide.


Figure JPOXMLDOC01-appb-T000031
Figure JPOXMLDOC01-appb-T000031
Figure JPOXMLDOC01-appb-T000032
Figure JPOXMLDOC01-appb-T000032
　表２４～２９の配列中、大文字は、式（ａ）で示されるＬＮＡ、式（ｂ）で示される２’－ＯＭｅ又は式（ｃ）で示されるアミドＢＮＡ（ＡｍＮＡ）のいずれかを表す。小文字はＤＮＡを表す。ヌクレオシド間の結合は、オリゴヌクレオチド全体を通してホスホロチオエート（Ｐ＝Ｓ）である。
In the sequences of Tables 24 to 29, capital letters represent either LNA represented by formula (a), 2′-OMe represented by formula (b), or amide BNA (AmNA) represented by formula (c). Lower case letters represent DNA. The linkage between nucleosides is phosphorothioate (P = S) throughout the oligonucleotide.
Figure JPOXMLDOC01-appb-T000033
Figure JPOXMLDOC01-appb-T000033
Figure JPOXMLDOC01-appb-T000034
Figure JPOXMLDOC01-appb-T000034
Figure JPOXMLDOC01-appb-T000035
Figure JPOXMLDOC01-appb-T000035
Figure JPOXMLDOC01-appb-T000036
Figure JPOXMLDOC01-appb-T000036
Figure JPOXMLDOC01-appb-T000037
Figure JPOXMLDOC01-appb-T000037
Figure JPOXMLDOC01-appb-T000038
Figure JPOXMLDOC01-appb-T000038
実施例３：インビトロモデル　細胞培養
　細胞を、以下に記載される適当な培地で培養し、３７℃、９５～９８％湿度及び５％ＣＯ２で維持した。
　ＨＬＥ：ヒト肝ガン由来細胞株ＨＬＥは、ＤＭＥＭ　Ｌｏｗ　Ｇｌｕｃｏｓｅ（Ｓｉｇｍａ）＋１０％ウシ胎児血清（ＦＢＳ）＋Ｐｅｎｉｃｉｌｌｉｎ（１００ｕｎｉｔｓ／ｍＬ）＋Ｓｔｒｅｐｔｏｍｙｃｉｎ（１００ｕｇ／ｍＬ）で培養した。
　Ｈｅｐａ１ｃ１ｃ７：マウス肝ガン由来細胞株Ｈｅｐａ１ｃ１ｃ７は、α－ＭＥＭ（Ｇｉｂｃｏ）＋１０％　ＦＢＳ＋Ａｎｔｉｂｉｏｔｉｃ Ａｎｔｉｍｙｃｏｔｉｃ Ｓｏｌｕｔｉｏｎ（１０ｍＬ／Ｌ）で培養した。 Example 3: In Vitro Model Cell Culture Cells were cultured in the appropriate media described below and maintained at 37 ° C., 95-98% humidity and 5% CO 2 .
HLE: Human hepatoma-derived cell line HLE was cultured in DMEM Low Glucose (Sigma) + 10% fetal bovine serum (FBS) + Penicillin (100 units / mL) + Streptomycin (100 ug / mL).
Hepa1c1c7: The mouse liver cancer-derived cell line Hepa1c1c7 was cultured in α-MEM (Gibco) + 10% FBS + Antibiotic Anticolytic Solution (10 mL / L).
実施例４：ＡＣＳＬ１に対するアンチセンスオリゴヌクレオチドの評価
（１）細胞導入試薬を用いた導入実験による評価
　実施例１及び２に記載の通り、設計・製造したアンチセンスオリゴヌクレオチドを用いて、マウスＨｅｐａ１ｃ１ｃ７細胞又はヒトＨＬＥ細胞でノックダウン実験を行った。
　マウスＨｅｐａ１ｃ１ｃ７細胞に対しＬｉｐｏｆｅｃｔａｍｉｎｅ ＲＮＡｉＭＡＸ試薬（ｉｎｖｉｔｒｏｇｅｎ）を用いて導入を行った。細胞培養液にアンチセンスオリゴヌクレオチドの最終濃度が２０ｎＭとなるように添加した。導入２４時間後にＦａｓｔｌａｎｅ(ＱＩＡＧＥＮ)、もしくは、ＣｅｌｌＡｍｐ　ＲＮＡ　Ｐｒｅｐ　Ｋｉｔ（Ｔａｋａｒａ）にて細胞を回収し、Ｏｎｅ　Ｓｔｅｐ　ＳＹＢＲ　ＰｒｉｍｅＳｃｒｉｐｔ　ＰＬＵＳ　ＲＴ－ＰＣＲ　Ｋｉｔ（Ｔａｋａｒａ）により定量的ＰＣＲを行った。内在性コントロールとしてＧａｐｄｈを使用した。
　マウスＡｃｓｌ１の発現量を測定するために使用したプライマー配列は、
Ｆｗプライマー：ＡＧＧＴＧＣＴＴＣＡＧＣＣＣＡＣＣＡＴＣ（配列番号５０７）；
Ｒｖプライマー：ＡＡＡＧＴＣＣＡＡＣＡＧＣＣＡＴＣＧＣＴＴＣ（配列番号５０８）
を用い、
　マウスＧａｐｄｈの発現量を測定するために使用したプライマー配列は、
Ｆｗプライマー：ＴＧＴＧＴＣＣＧＴＣＧＴＧＧＡＴＣＴＧＡ（配列番号５０９）；
Ｒｖプライマー：ＴＴＧＣＴＧＴＴＧＡＡＧＴＣＧＣＡＧＧＡＧ（配列番号５１０）
を用いた。
　ヒトＨＬＥ細胞に対しＬｉｐｏｆｅｃｔａｍｉｎｅ ＬＴＸ試薬(ｉｎｖｉｔｒｏｇｅｎ)を用いて導入を行った。細胞培養液にアンチセンスオリゴヌクレオチドの最終濃度が２０ｎＭとなるように添加した。導入２４時間後にＦａｓｔｌａｎｅ(ＱＩＡＧＥＮ)、もしくは、ＣｅｌｌＡｍｐ　ＲＮＡ　Ｐｒｅｐ　Ｋｉｔ（Ｔａｋａｒａ）にて細胞を回収し、Ｏｎｅ　Ｓｔｅｐ　ＳＹＢＲ　ＰｒｉｍｅＳｃｒｉｐｔ　ＰＬＵＳ　ＲＴ－ＰＣＲ　Ｋｉｔ（Ｔａｋａｒａ）により定量的ＰＣＲを行った。内在性コントロールとしてＧＡＰＤＨを使用した。
　ヒトＡＣＳＬ１の発現量を測定するために使用したプライマー配列は、
Ｆｗプライマー：ＧＣＡＧＣＧＧＣＡＴＣＡＴＣＡＧＡＡＡＣ（配列番号５１７）；
Ｒｖプライマー：ＴＧＴＣＡＣＣＡＴＣＡＧＣＣＧＧＡＣＴＣ（配列番号５１８）
を用い、
　ヒトＧＡＰＤＨの発現量を測定するために使用したプライマー配列は、
Ｆｗプライマー：ＧＣＡＣＣＧＴＣＡＡＧＧＣＴＧＡＧＡＡＣ（配列番号５１９）；
Ｒｖプライマー：ＴＧＧＴＧＡＡＧＡＣＧＣＣＡＧＴＧＧＡ（配列番号５２０）
を用いた。
　結果を表３０～３２に示す。表３０にはＬｉｐｏｆｅｃｔａｍｉｎｅ ＲＮＡｉＭＡＸ試薬を用いて細胞導入したアンチセンスオリゴヌクレオチドに関し、Ｇａｐｄｈで正規化したマウスＨｅｐａ１ｃ１ｃ７細胞におけるＡｃｓｌ１のｍＲＮＡ減少量を、未処理細胞に対する割合をノックダウン効率として示した。表３１又は表３２にはＬｉｐｏｆｅｃｔａｍｉｎｅ ＬＴＸ試薬を用いて細胞導入したアンチセンスオリゴヌクレオチドに関し、ＧＡＰＤＨで正規化したヒトＨＬＥ細胞におけるＡＣＳＬ１のｍＲＮＡ減少量を、未処理細胞に対する割合をノックダウン効率として示した。
　この結果、本発明アンチセンスオリゴヌクレオチドは、他のアンチセンスオリゴヌクレオチドと比較して、マウスＡｃｓｌ１又はヒトＡＣＳＬ１に対して高いノックダウン活性を示した。 Example 4: Evaluation of antisense oligonucleotides against ACSL1 (1) Evaluation by introduction experiment using cell introduction reagent Mouse Hepa1c1c7 cells using antisense oligonucleotides designed and manufactured as described in Examples 1 and 2 Alternatively, knockdown experiments were performed with human HLE cells.
Introduction into mouse Hepa1c1c7 cells was performed using Lipofectamine RNAiMAX reagent (invitrogen). The final concentration of antisense oligonucleotide was added to the cell culture solution to 20 nM. 24 hours after the introduction, cells were collected with Fastlane (QIAGEN) or CellAmp RNA Prep Kit (Takara), and quantitative PCR was performed with One Step SYBR PrimeScript PLUS RT-PCR Kit (Takara). Gapdh was used as an endogenous control.
The primer sequence used to measure the expression level of mouse Acsl1 is:
Fw primer: AGGTGCTTCAGCCCACATC (SEQ ID NO: 507);
Rv primer: AAAGTCCAACAGCCATCGCTTC (SEQ ID NO: 508)
Use
The primer sequence used to measure the expression level of mouse Gapdh is:
Fw primer: TGTGTCCGTCGTGGATCTGA (SEQ ID NO: 509);
Rv primer: TTGCTGTTTGAAGTCGCAGAG (SEQ ID NO: 510)
Was used.
Human HLE cells were introduced using Lipofectamine LTX reagent (invitrogen). The final concentration of antisense oligonucleotide was added to the cell culture solution to 20 nM. 24 hours after the introduction, cells were collected with Fastlane (QIAGEN) or CellAmp RNA Prep Kit (Takara), and quantitative PCR was performed with One Step SYBR PrimeScript PLUS RT-PCR Kit (Takara). GAPDH was used as an endogenous control.
The primer sequence used to measure the expression level of human ACSL1 is
Fw primer: GCAGCGGGCATCATCAGAAAC (SEQ ID NO: 517);
Rv primer: TGTCACATCATAGCCCGACTC (SEQ ID NO: 518)
Use
The primer sequence used to measure the expression level of human GAPDH is:
Fw primer: GCACCGTCAAGGCTGGAAC (SEQ ID NO: 519);
Rv primer: TGGTGAAGACGCCAGTGGA (SEQ ID NO: 520)
Was used.
The results are shown in Tables 30 to 32. Table 30 shows the amount of Acsl1 mRNA decreased in mouse Hepa1c1c7 cells normalized with Gapdh as knockdown efficiency for antisense oligonucleotides introduced into cells using Lipofectamine RNAiMAX reagent. Table 31 or Table 32 shows the amount of decrease in ACSL1 mRNA in human HLE cells normalized with GAPDH as knockdown efficiency for antisense oligonucleotides introduced into cells using Lipofectamine LTX reagent. .
As a result, the antisense oligonucleotide of the present invention showed high knockdown activity against mouse Acsl1 or human ACSL1 compared to other antisense oligonucleotides.
（試験１：Ｈｅｐａ１ｃ１ｃ７細胞）





Figure JPOXMLDOC01-appb-T000039
 (Test 1: Hepa1c1c7 cells)





Figure JPOXMLDOC01-appb-T000039
（試験２：ＨＬＥ細胞）
Figure JPOXMLDOC01-appb-T000040
 　 (Test 2: HLE cells)
Figure JPOXMLDOC01-appb-T000040
（試験３：ＨＬＥ細胞）
Figure JPOXMLDOC01-appb-T000041
 (Test 3: HLE cells)
Figure JPOXMLDOC01-appb-T000041
　次に、他のＡＣＳＬファミリー（ＡＣＳＬ３，ＡＣＳＬ４，ＡＣＳＬ５）との交差性を定量的ＰＣＲにより評価した。内在性コントロールとしてＧＡＰＤＨを使用した。
　マウスＡｃｓｌ３の発現量を測定するために使用したプライマー配列は、
Ｆｗプライマー：ＧＣＡＡＣＡＡＣＧＣＡＧＣＧＡＴＴＣＡ（配列番号５１１）；
Ｒｖプライマー：ＡＧＣＡＡＡＣＴＡＡＴＧＧＴＧＣＴＣＣＣＡＣＴＣ（配列番号５１２）
を用いた。
　マウスＡｃｓｌ４の発現量を測定するために使用したプライマー配列は、
Ｆｗプライマー：ＧＣＣＡＴＧＧＡＡＧＣＴＧＡＡＡＴＡＣＴＧＡＡＡＧ（配列番号５１３）；
Ｒｖプライマー：ＧＡＡＧＧＣＡＴＣＴＧＴＴＡＣＣＡＡＡＣＣＡＧＴＣ（配列番号５１４）
を用いた。
　マウスＡｃｓｌ５の発現量を測定するために使用したプライマー配列は、
Ｆｗプライマー：ＣＡＴＴＣＧＧＣＧＧＧＡＣＡＧＴＴＴＧ（配列番号５１５）；
Ｒｖプライマー：ＡＴＣＣＣＡＴＴＧＣＡＧＣＣＣＴＧＡＡＧ（配列番号５１６）
を用いた。
　ヒトＡＣＳＬ３の発現量を測定するために使用したプライマー配列は、
Ｆｗプライマー：ＡＴＡＣＧＧＧＣＴＣＡＣＴＧＡＡＴＣＴＧＣＴＧ（配列番号５２１）；
Ｒｖプライマー：ＡＧＣＡＡＡＣＴＡＡＴＧＧＴＧＣＴＣＣＣＡＣＴＣ（配列番号５２２）
を用いた。
　ヒトＡＣＳＬ４の発現量を測定するために使用したプライマー配列は、
Ｆｗプライマー：ＧＡＡＴＧＧＡＴＧＡＴＴＧＣＡＧＣＡＣＡＧＡ（配列番号５２３）；
Ｒｖプライマー：ＣＣＴＣＡＧＡＴＴＣＡＴＴＴＡＧＣＣＣＡＴＧＡＡＣ（配列番号５２４）
を用いた。
ヒトＡＣＳＬ５の発現量を測定するために使用したプライマー配列は、
Ｆｗプライマー：ＧＧＡＡＣＴＣＴＧＡＡＧＡＴＣＡＴＣＧＡＣＣＧＴＡ（配列番号５２５）；
Ｒｖプライマー：ＣＴＧＴＧＴＣＡＧＧＡＡＣＣＡＣＣＡＣＴＣＣＴＡ（配列番号５２６）
を用いた。 Next, cross-reactivity with other ACSL families (ACSL3, ACSL4, ACSL5) was evaluated by quantitative PCR. GAPDH was used as an endogenous control.
The primer sequence used to measure the expression level of mouse Acsl3 is:
Fw primer: GCAACAACGCAGCGATTCA (SEQ ID NO: 511);
Rv primer: AGCAAACTAATGGTGCCTCCACTC (SEQ ID NO: 512)
Was used.
The primer sequence used to measure the expression level of mouse Acsl4 is:
Fw primer: GCCATGGAAGCTGAAATACGAAAG (SEQ ID NO: 513);
Rv primer: GAAGGGCATCTGTTACAAAACCAGTC (SEQ ID NO: 514)
Was used.
The primer sequence used to measure the expression level of mouse Acsl5 is:
Fw primer: CATTCGGCGGGACAGTTTG (SEQ ID NO: 515);
Rv primer: ATCCCATTGCCAGCCCCTGAAG (SEQ ID NO: 516)
Was used.
The primer sequence used to measure the expression level of human ACSL3 is:
Fw primer: ATACGGGCTCACTGAATCTGCTG (SEQ ID NO: 521);
Rv primer: AGCAAACTAATGGTGCCTCCACTC (SEQ ID NO: 522)
Was used.
The primer sequence used to measure the expression level of human ACSL4 is
Fw primer: GAATGGATGATGTCAGCACAGA (SEQ ID NO: 523);
Rv primer: CCTCAGATTCATTTCCCCCATGAAC (SEQ ID NO: 524)
Was used.
The primer sequence used to measure the expression level of human ACSL5 is
Fw primer: GGAACTCTGAAGATCATCGACCGTA (SEQ ID NO: 525);
Rv primer: CGTTGTCAGGAACCACCACTCCTA (SEQ ID NO: 526)
Was used.
　結果を表３３～３４に示す。各表には、ＧＡＰＤＨで正規化したＡＣＳＬ３、ＡＣＳＬ４又はＡＣＳＬ５のｍＲＮＡ減少量について、未処理細胞に対する割合をノックダウン効率として示した。表中のＮ．Ｄ．は、ＡＣＳＬ３、ＡＣＳＬ４又はＡＣＳＬ５のｍＲＮＡ減少量が検出限界以下である、あるいはｍＲＮＡ量を増加させており、ＡＣＳＬ３、ＡＣＳＬ４又はＡＣＳＬ５を抑制していないことを意味する。
　この結果、本発明アンチセンスオリゴヌクレオチドは、他のＡＣＳＬファミリー（ＡＣＳＬ３，ＡＣＳＬ４，ＡＣＳＬ５）を５０％以上ノックダウンせず、ＡＣＳＬ１に対し高い特異性を示した。 The results are shown in Tables 33-34. In each table, the ratio of the decrease in mRNA of ACSL3, ACSL4, or ACSL5 normalized by GAPDH to the untreated cells is shown as the knockdown efficiency. N. in the table. D. Means that the amount of mRNA decrease in ACSL3, ACSL4 or ACSL5 is below the detection limit, or the amount of mRNA is increased, and ACSL3, ACSL4 or ACSL5 is not suppressed.
As a result, the antisense oligonucleotide of the present invention did not knock down other ACSL families (ACSL3, ACSL4, ACSL5) by 50% or more and showed high specificity for ACSL1.
（試験４：Ｈｅｐａ１ｃ１ｃ７細胞）
Figure JPOXMLDOC01-appb-T000042
 (Test 4: Hepa1c1c7 cells)
Figure JPOXMLDOC01-appb-T000042
（試験５：ＨＬＥ細胞）
Figure JPOXMLDOC01-appb-T000043
（２）自然取り込みによる導入実験
　実施例１及び２に記載の通り、設計・製造したアンチセンスオリゴヌクレオチドを細胞へ導入した。マウスＡｃｓｌ１に対するアンチセンスオリゴヌクレオチドはマウスＨｅｐａ１ｃ１ｃ７細胞へ導入を行い、ヒトＡＣＳＬ１に対するアンチセンスオリゴヌクレオチドはヒトＨＬＥ細胞へ導入を行った。
　自然取り込みによる導入実験では、すべての細胞において、試薬を用いずにアンチセンスオリゴヌクレオチドを細胞へ導入した。マウスＨｅｐａ１ｃ１ｃ７及びヒトＨＬＥ細胞において、導入１２０時間後にＦａｓｔｌａｎｅ(ＱＩＡＧＥＮ)、もしくは、ＣｅｌｌＡｍｐ　ＲＮＡ　Ｐｒｅｐ　Ｋｉｔ（Ｔａｋａｒａ）にて細胞を回収し、Ｏｎｅ　Ｓｔｅｐ　ＳＹＢＲ　ＰｒｉｍｅＳｃｒｉｐｔ　ＰＬＵＳ　ＲＴ－ＰＣＲ　Ｋｉｔ（Ｔａｋａｒａ）により定量的ＰＣＲを行った。内在性コントロールとしてＧＡＰＤＨを使用した。
　マウスＡｃｓｌ１及びマウスＧａｐｄｈの発現量を測定するために使用したプライマー配列は、上記（１）で示したものを用いた。
　ヒトＡＣＳＬ１の発現量を測定するために使用したプライマー配列は、
Ｆｗプライマー：ＧＣＡＧＣＧＧＣＡＴＣＡＴＣＡＧＡＡＡＣ（配列番号５１７）；
Ｒｖプライマー：ＴＧＴＣＡＣＣＡＴＣＡＧＣＣＧＧＡＣＴＣ（配列番号５１８）
を用い、
　ヒトＧＡＰＤＨの発現量を測定するために使用したプライマー配列は、
Ｆｗプライマー：ＧＣＡＣＣＧＴＣＡＡＧＧＣＴＧＡＧＡＡＣ（配列番号５１９）；
Ｒｖプライマー：ＴＧＧＴＧＡＡＧＡＣＧＣＣＡＧＴＧＧＡ（配列番号５２０）
を用いた。 (Test 5: HLE cells)
Figure JPOXMLDOC01-appb-T000043
(2) Introduction experiment by natural uptake As described in Examples 1 and 2, designed and produced antisense oligonucleotides were introduced into cells. An antisense oligonucleotide for mouse Acsl1 was introduced into mouse Hepa1c1c7 cells, and an antisense oligonucleotide for human ACSL1 was introduced into human HLE cells.
In the introduction experiment by natural uptake, in all cells, antisense oligonucleotides were introduced into cells without using reagents. In mouse Hepa1c1c7 and human HLE cells, cells were collected with Fastlane (QIAGEN) or CellAmp RNA Prep Kit (Takara) 120 hours after transfection, and quantitative analysis was performed by One Step SYBR PrimeScript PLUS RT-PCR Kit (Takara). went. GAPDH was used as an endogenous control.
The primer sequences used for measuring the expression levels of mouse Acsl1 and mouse Gapdh were those shown in (1) above.
The primer sequence used to measure the expression level of human ACSL1 is
Fw primer: GCAGCGGGCATCATCAGAAAC (SEQ ID NO: 517);
Rv primer: TGTCACATCATAGCCCGACTC (SEQ ID NO: 518)
Use
The primer sequence used to measure the expression level of human GAPDH is:
Fw primer: GCACCGTCAAGGCTGGAAC (SEQ ID NO: 519);
Rv primer: TGGTGAAGACGCCAGTGGA (SEQ ID NO: 520)
Was used.
　結果を表３５～４６に示す。各表には、ＧＡＰＤＨで正規化したＡＣＳＬ１のｍＲＮＡ減少量を、未処理細胞に対する割合をノックダウン効率として示した。この結果、本発明アンチセンスオリゴヌクレオチドは、Ｈｅｐａ１ｃ１ｃ７細胞及び／又はＨＬＥ細胞において、マウスＡｃｓｌ１及び/又はヒトＡＣＳＬ１に対して高いノックダウン活性を示した。 Results are shown in Tables 35-46. In each table, the amount of decrease in ACSL1 mRNA normalized by GAPDH is shown as the ratio of untreated cells as knockdown efficiency. As a result, the antisense oligonucleotide of the present invention showed high knockdown activity against mouse Acsl1 and / or human ACSL1 in Hepa1c1c7 cells and / or HLE cells.
（試験１－１: Ｈｅｐａ１ｃ１ｃ７細胞）
Figure JPOXMLDOC01-appb-T000044
 (Test 1-1: Hepa1c1c7 cells)
Figure JPOXMLDOC01-appb-T000044
（試験１－２： Ｈｅｐａ１ｃ１ｃ７細胞）
Figure JPOXMLDOC01-appb-T000045
 (Test 1-2: Hepa1c1c7 cells)
Figure JPOXMLDOC01-appb-T000045
（試験１－３：ＨＬＥ細胞）
Figure JPOXMLDOC01-appb-T000046
 (Test 1-3: HLE cells)
Figure JPOXMLDOC01-appb-T000046
（試験１－４：ＨＬＥ細胞）
Figure JPOXMLDOC01-appb-T000047
 (Test 1-4: HLE cells)
Figure JPOXMLDOC01-appb-T000047
（試験１－５： Ｈｅｐａ１ｃ１ｃ７細胞）
Figure JPOXMLDOC01-appb-T000048
 (Test 1-5: Hepa1c1c7 cells)
Figure JPOXMLDOC01-appb-T000048
（試験１－６： Ｈｅｐａ１ｃ１ｃ７細胞）
Figure JPOXMLDOC01-appb-T000049
 (Test 1-6: Hepa1c1c7 cells)
Figure JPOXMLDOC01-appb-T000049
（試験１－７： Ｈｅｐａ１ｃ１ｃ７細胞）

Figure JPOXMLDOC01-appb-T000050
 (Test 1-7: Hepa1c1c7 cells)

Figure JPOXMLDOC01-appb-T000050
（試験１－８： Ｈｅｐａ１ｃ１ｃ７細胞）
Figure JPOXMLDOC01-appb-T000051
 (Test 1-8: Hepa1c1c7 cells)
Figure JPOXMLDOC01-appb-T000051
（試験１－９： Ｈｅｐａ１ｃ１ｃ７細胞）
Figure JPOXMLDOC01-appb-T000052
 (Test 1-9: Hepa1c1c7 cells)
Figure JPOXMLDOC01-appb-T000052
（試験１－１０：ＨＬＥ細胞）
Figure JPOXMLDOC01-appb-T000053
 (Test 1-10: HLE cells)
Figure JPOXMLDOC01-appb-T000053
（試験１－１１：ＨＬＥ細胞）
Figure JPOXMLDOC01-appb-T000054
 (Test 1-11: HLE cells)
Figure JPOXMLDOC01-appb-T000054
（試験１－１２：ＨＬＥ細胞）
Figure JPOXMLDOC01-appb-T000055
 (Test 1-12: HLE cells)
Figure JPOXMLDOC01-appb-T000055
　次に、他のＡＣＳＬファミリー（ＡＣＳＬ３，ＡＣＳＬ４，ＡＣＳＬ５）との交差性を定量的ＰＣＲにより評価した。内在性コントロールとしてＧＡＰＤＨを使用した。
　マウスＡｃｓｌ３、マウスＡｃｓｌ４、マウスＡｃｓｌ５、ヒトＡＣＳＬ３、ヒトＡＣＤＬ４又はヒトＡＣＳＬ５の発現量を測定するために使用したプライマー配列は、上記（１）で示したものを用いた。 Next, cross-reactivity with other ACSL families (ACSL3, ACSL4, ACSL5) was evaluated by quantitative PCR. GAPDH was used as an endogenous control.
The primer sequence used for measuring the expression level of mouse Acsl3, mouse Acsl4, mouse Acsl5, human ACSL3, human ACDL4 or human ACSL5 was the one shown in (1) above.
　結果を表４７～５２に示す。各表には、ＧＡＰＤＨで正規化したＡＣＳＬ３、ＡＣＳＬ４又はＡＣＳＬ５のｍＲＮＡ減少量について、未処理細胞に対する割合をノックダウン効率として示した。表中のＮ．Ｄ．は、ＡＣＳＬ３、ＡＣＳＬ４又はＡＣＳＬ５のｍＲＮＡ減少量が検出限界以下である、あるいはｍＲＮＡ量を増加させており、ＡＣＳＬ３、ＡＣＳＬ４又はＡＣＳＬ５を抑制していないことを意味する。
　この結果、本発明アンチセンスオリゴヌクレオチドは、５μＭで細胞培養液へ添加した場合でも他のＡＣＳＬファミリー（ＡＣＳＬ３，ＡＣＳＬ４，ＡＣＳＬ５）を５０％以上ノックダウンせず、ＡＣＳＬ１に対して高い特異性を示した。 The results are shown in Tables 47-52. In each table, the ratio of the decrease in mRNA of ACSL3, ACSL4, or ACSL5 normalized by GAPDH to the untreated cells is shown as the knockdown efficiency. N. in the table. D. Means that the amount of mRNA decrease in ACSL3, ACSL4 or ACSL5 is below the detection limit, or the amount of mRNA is increased, and ACSL3, ACSL4 or ACSL5 is not suppressed.
As a result, the antisense oligonucleotide of the present invention does not knock down other ACSL families (ACSL3, ACSL4, ACSL5) more than 50% even when added to the cell culture medium at 5 μM, and shows high specificity to ACSL1. It was.
（試験２－１：Ｈｅｐａ１ｃ１ｃ７細胞）

Figure JPOXMLDOC01-appb-T000056














Figure JPOXMLDOC01-appb-T000057
 (Test 2-1: Hepa1c1c7 cells)

Figure JPOXMLDOC01-appb-T000056














Figure JPOXMLDOC01-appb-T000057
（試験２－２：ＨＬＥ細胞）
Figure JPOXMLDOC01-appb-T000058
 (Test 2-2: HLE cells)
Figure JPOXMLDOC01-appb-T000058
（試験２－３：Ｈｅｐａ１ｃ１ｃ７細胞）
Figure JPOXMLDOC01-appb-T000059
 (Test 2-3: Hepa1c1c7 cells)
Figure JPOXMLDOC01-appb-T000059
（試験２－４：ＨＬＥ細胞）
Figure JPOXMLDOC01-appb-T000060
 (Test 2-4: HLE cells)
Figure JPOXMLDOC01-appb-T000060
（試験２－５：ＨＬＥ細胞）
Figure JPOXMLDOC01-appb-T000061
 (Test 2-5: HLE cells)
Figure JPOXMLDOC01-appb-T000061
　実施例２の表において、標的部位のカラム中にマウス及びヒトの標的部位が記載されている配列は、それぞれヒトとマウスのＡＣＳＬ１遺伝子の同部分に対する配列として設計している。ヒトとマウスにおいて同一の配列であっても、共に特異的かつ高いノックダウン活性を示すとは限らないことから、共に特異的かつ高いノックダウン活性を示した本発明のアンチセンスオリゴヌクレオチドは、創薬上非常に有用である。 In the table of Example 2, the sequences in which the mouse and human target sites are described in the target site column are designed as sequences for the same part of the human and mouse ACSL1 gene, respectively. Even if the sequences are the same in human and mouse, they do not always show specific and high knockdown activity. Therefore, the antisense oligonucleotide of the present invention showing both specific and high knockdown activity is Medicinal very useful.
実施例５：アンチセンスオリゴヌクレオチド単回投与時のｉｎ　ｖｉｖｏ活性評価
　Ｃ５７ＢＬ／６Ｊ（オス１０週齢, 日本クレア）に、生理食塩水（大塚生食注、大塚製薬工場）に溶解した本発明のアンチセンスオリゴヌクレオチド溶液、約０．２ｍＬを、マウス個体あたり投与量が１０ｍｇ／ｋｇ又は２０ｍｇ／ｋｇとなるように皮下投与した。投与後３日、７日、及び１４日後にソムノペンチル麻酔下で全血約０．５ｍＬ及び肝臓組織を採取した。肝臓からのＲＮＡ抽出はＲＮｅａｓｙ　９６　Ｕｎｉｖｅｒｓａｌ　Ｔｉｓｓｕｅ　Ｋｉｔ　（Ｑｉａｇｅｎ社製)を用いてメーカー推奨プロトコル通りに行った。得られたＲＮＡのうち１０００ｎｇを、ＳｕｐｅｒＳｃｒｉｐｔ　ＩＩＩ　Ｆｉｒｓｔ－Ｓｔｒａｎｄ　Ｓｙｎｔｈｅｓｉｓ　ＳｕｐｅｒＭｉｘ　ｆｏｒ　ｑＲＴ－ＰＣＲ　（Ｌｉｆｅ　Ｓｃｉｅｎｃｅ社製)を用いて、標準プロトコルに準じて逆転写を行うことでｃＤＮＡを得た。ＳＹＢＲ　Ｐｒｅｍｉｘ　Ｅｘ　Ｔａｑ　ＩＩ（タカラバイオ社製）を用いて定量的ＰＣＲを行った。内在性コントロールとしてＧＡＰＤＨを使用し、プライマーは実施例４と同一のものを用いた。
　結果を表５３、５４に示す。各表には、ＧＡＰＤＨで正規化したＡＣＳＬ１のｍＲＮＡ量について、生理食塩水投与群に対する割合をノックダウン効率として示した。この結果、本発明アンチセンスオリゴヌクレオチドは、ｉｎ　ｖｉｖｏにおいても濃度依存的なノックダウン活性を示すことが確認できた。 Example 5: Evaluation of in vivo activity at the time of a single administration of an antisense oligonucleotide C57BL / 6J (male, 10 weeks old, CLEA, Japan) was dissolved in physiological saline (Otsuka raw food injection, Otsuka Pharmaceutical Factory). About 0.2 mL of the sense oligonucleotide solution was subcutaneously administered so that the dose per mouse individual was 10 mg / kg or 20 mg / kg. About 0.5 mL of whole blood and liver tissue were collected under somnenopentyl anesthesia 3 days, 7 days, and 14 days after administration. RNA extraction from the liver was performed according to the manufacturer's recommended protocol using RNeasy 96 Universal Tissue Kit (Qiagen). 1000 ng of the obtained RNA was reverse-transcribed according to a standard protocol using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (manufactured by Life Science) to obtain cDNA. Quantitative PCR was performed using SYBR Premix Ex Taq II (manufactured by Takara Bio Inc.). GAPDH was used as an endogenous control, and the same primers as in Example 4 were used.
The results are shown in Tables 53 and 54. In each table, the ratio of the ACSL1 mRNA amount normalized by GAPDH to the physiological saline administration group is shown as the knockdown efficiency. As a result, it was confirmed that the antisense oligonucleotide of the present invention showed a concentration-dependent knockdown activity even in vivo.
Figure JPOXMLDOC01-appb-T000062
Figure JPOXMLDOC01-appb-T000062
Figure JPOXMLDOC01-appb-T000063
Figure JPOXMLDOC01-appb-T000063
実施例６：アンチセンスオリゴ反復投与時のｉｎ　ｖｉｖｏ活性評価
　Ｃ５７ＢＬ／６Ｊ（オス７週齢, 日本クレア）に４週間、高脂脂肪食（６０％ｋｃａｌ脂肪：ＴｅｓｔＤｉｅｔ社製）を４週間与えることで食餌誘導性肥満（ＤＩＯ）マウスを作製した。ＤＩＯマウスへ、生理食塩水（大塚生食注、大塚製薬工場）に溶解した本発明のアンチセンスオリゴヌクレオチド溶液、約０．２ｍＬを毎週１回皮下投与した。用量は、マウス個体あたり投与量が５ｍｇ／ｋｇ／週、１０ｍｇ／ｋｇ／週、及び２０ｍｇ／ｋｇ／週となるようにし、２週にわたり週１回投与した。初回投与１４日後にソムノペンチル麻酔下で全血約０．５ｍＬ及び肝臓組織を採取し、活性評価及び肝臓毒性の評価を行った。
　結果を表５５に示す。該表には、ＧＡＰＤＨで正規化したＡＣＳＬ１のｍＲＮＡ量について、生理食塩水投与群に対する割合をノックダウン効率（ＫＤ）として示した。また、肝臓毒性評価をＡＬＴ（アラニンアミノトランスフェラーゼ）の量で示した。
この結果、本発明のアンチセンスオリゴヌクレオチドは、ｉｎ　ｖｉｖｏで反復投与した際にも濃度依存的なノックダウン活性を示すことが確認できた。さらに、本発明のアンチセンスオリゴヌクレオチドが肝臓毒性を示さないことも確認できた。本発明のアンチセンスオリゴヌクレオチドは、肝臓のＡＣＳＬ１の発現を抑制することによって体重の増加の抑制及び血糖値を降下させるため、肝臓での働きにより肝臓毒性を示さないことは、創薬上非常に有用である。 Example 6: Evaluation of in vivo activity at the time of repeated administration of antisense oligo C57BL / 6J (male 7-week-old, Claire, Japan) is given a high fat fat diet (60% kcal fat: manufactured by TestDiet) for 4 weeks. To produce diet-induced obese (DIO) mice. About 0.2 mL of the antisense oligonucleotide solution of the present invention dissolved in physiological saline (Otsuka raw food injection, Otsuka Pharmaceutical Factory) was subcutaneously administered once weekly to DIO mice. The dose was 5 mg / kg / week, 10 mg / kg / week, and 20 mg / kg / week, and the dose was administered once a week for 2 weeks. 14 days after the first administration, about 0.5 mL of whole blood and liver tissue were collected under somnopentyl anesthesia, and activity evaluation and liver toxicity evaluation were performed.
The results are shown in Table 55. In this table, the ratio of ACSL1 mRNA amount normalized by GAPDH to the physiological saline administration group is shown as knockdown efficiency (KD). In addition, liver toxicity evaluation was shown by the amount of ALT (alanine aminotransferase).
As a result, it was confirmed that the antisense oligonucleotide of the present invention showed a concentration-dependent knockdown activity even after repeated administration in vivo. Furthermore, it was confirmed that the antisense oligonucleotide of the present invention did not show liver toxicity. The antisense oligonucleotide of the present invention suppresses the increase in body weight and suppresses blood glucose level by suppressing the expression of ACSL1 in the liver. Therefore, it does not exhibit liver toxicity due to its action in the liver. Useful.
Figure JPOXMLDOC01-appb-T000064
Figure JPOXMLDOC01-appb-T000064
実施例７：アンチセンスオリゴ頻回投与時のｉｎ　ｖｉｖｏ活性評価
　Ｃ５７ＢＬ／６Ｊ（オス７週齢, 日本クレア）に４週間、高脂脂肪食（６０％ｋｃａｌ脂肪：ＴｅｓｔＤｉｅｔ社製）を４週間与えることで食餌誘導性肥満（ＤＩＯ）マウスを作製した。ＤＩＯマウスへ、生理食塩水（大塚生食注、大塚製薬工場）に溶解した本発明のアンチセンスオリゴヌクレオチド溶液、約０．２ｍＬを毎週１回皮下投与した。用量は、マウス個体あたり投与量が１０ｍｇ／ｋｇ／週及び２０ｍｇ／ｋｇ週となるようにし、４週間投与を継続した。初回投与後２８日で血漿及び肝臓を摘出し、ｍＲＮＡとタンパク質量の発現変化について調べた。
　結果を表５６に示す。該表には、ＧＡＰＤＨで正規化したＡＣＳＬ１のｍＲＮＡ量について、生理食塩水投与群に対する割合をノックダウン効率として示した。この結果、本発明のアンチセンスヌクレオチドは、ｉｎ　ｖｉｖｏで頻回投与した際にも濃度依存的なノックダウン活性を示すことが確認できた。
　さらに、タンパク質量の発現変化についての結果を図１に示す。この結果、本発明のアンチセンスオリゴヌクレオチドは、ｉｎ　ｖｉｖｏで頻回投与した際にも、タンパク質レベルでＡＣＳＬ１をノックダウン可能であることが確認できた。またｍＲＮＡとタンパク質のノックダウン効率が概ね一致することを見出した。 Example 7: Evaluation of in vivo activity at the time of frequent administration of antisense oligo C57BL / 6J (male 7 weeks old, Claire, Japan) is given 4 weeks and a high fat fat diet (60% kcal fat: manufactured by TestDiet) for 4 weeks Thus, diet-induced obese (DIO) mice were produced. About 0.2 mL of the antisense oligonucleotide solution of the present invention dissolved in physiological saline (Otsuka raw food injection, Otsuka Pharmaceutical Factory) was subcutaneously administered once weekly to DIO mice. The dose was 10 mg / kg / week and 20 mg / kg week per individual mouse, and the administration was continued for 4 weeks. At 28 days after the first administration, plasma and liver were excised and examined for changes in mRNA and protein expression.
The results are shown in Table 56. The table shows the ratio of the ACSL1 mRNA amount normalized with GAPDH to the physiological saline administration group as the knockdown efficiency. As a result, it was confirmed that the antisense nucleotide of the present invention exhibited a concentration-dependent knockdown activity even when administered frequently in vivo.
Furthermore, the result about the expression change of protein amount is shown in FIG. As a result, it was confirmed that the antisense oligonucleotide of the present invention can knock down ACSL1 at the protein level even when it is frequently administered in vivo. Moreover, it discovered that knockdown efficiency of mRNA and protein was in general agreement.
Figure JPOXMLDOC01-appb-T000065
Figure JPOXMLDOC01-appb-T000065
実施例８：アンチセンスオリゴ反復投与時の体重増加抑制効果
　Ｃ５７ＢＬ／６Ｊ（オス７週齢, 日本クレア）に４週間、高脂脂肪食（６０％ｋｃａｌ脂肪：ＴｅｓｔＤｉｅｔ社製）を４週間与えることで食餌誘導性肥満（ＤＩＯ）マウスを作製した。体重の平均値が同じになるように一群７～８匹に分け、生理食塩水（大塚生食注、大塚製薬工場）に溶解した本発明のアンチセンスオリゴヌクレオチド溶液、約０．２ｍＬを毎週１回皮下投与した。用量は、マウス個体あたり投与量が１０ｍｇ／ｋｇ／週、及び２０ｍｇ／ｋｇとなるようにし、４週間投与を継続した。体重推移を図２、摂餌量の変化を図３に示す。生理食塩水投与群と比較して、本発明のアンチセンスオリゴヌクレオチド投与群はいずれも摂餌量に顕著な変化は見られなかったが、本発明のアンチセンスオリゴヌクレオチドを投与した群ではいずれも体重増加抑制が確認された。この結果から、肝臓中のＡＣＳＬ１発現をアンチセンスオリゴで抑制することで、抗肥満効果が確認された。 Example 8: Effect of inhibiting weight gain upon repeated administration of antisense oligo C57BL / 6J (male 7-week-old, Claire, Japan) is given a high fat fat diet (60% kcal fat: manufactured by TestDiet) for 4 weeks. To produce diet-induced obese (DIO) mice. Divide the group into 7 to 8 animals so that the average value of the body weight is the same. About 0.2 mL of the antisense oligonucleotide solution of the present invention dissolved in physiological saline (Otsuka raw food injection, Otsuka Pharmaceutical Factory) once a week. It was administered subcutaneously. The dose was adjusted to 10 mg / kg / week and 20 mg / kg per mouse individual, and the administration was continued for 4 weeks. The weight transition is shown in FIG. 2, and the change in food intake is shown in FIG. Compared with the physiological saline administration group, none of the antisense oligonucleotide administration groups of the present invention showed a significant change in food intake, but none of the groups administered with the antisense oligonucleotide of the present invention. Inhibition of weight gain was confirmed. From this result, the anti-obesity effect was confirmed by suppressing the ACSL1 expression in the liver with an antisense oligo.
実施例９：アンチセンスオリゴ単回投与時の肝臓毒性評価
本発明のアンチセンスオリゴヌクレオチドに対して、マウス肝臓における毒性評価を行った。具体的には、血漿中に含まれるアスパラギン酸アミノトランスフェラーゼ（ＡＳＴ）及びアラニンアミノトランスフェラーゼ（ＡＬＴ）を肝臓毒性のマーカーとして測定した。Ｃ５７ＢＬ／６Ｊ（オス１０週齢, 日本クレア）に、生理食塩水（大塚生食注、大塚製薬工場）に溶解したアンチセンスオリゴ溶液、約０．２ｍＬを、マウス個体あたり投与量が１０ｍｇ／ｋｇ、２０ｍｇ／ｋｇとなるように皮下投与し、投与後３日、７日、及び１４日後にソムノペンチル麻酔下で全血約０．５ｍＬを採取し、直ちにヘパリン（味の素製薬社製）を最終濃度０．０１Ｕ/μｌとなるように添加した。得られた血漿中のＡＳＴ及びＡＬＴを、トランスアミナーゼＣ２キットワコー（和光純薬工業社製）を用い、添付のマニュアルに従って測定した。
結果を表５７～５９に示す。この結果、本発明のアンチセンスオリゴヌクレオチドを投与したマウス血中において、生理食塩水投与群と比べて有意なＡＳＴ及びＡＬＴの上昇は観察されず、本発明のアンチセンスオリゴヌクレオチドが肝臓毒性を示さないことが確認できた。本発明のアンチセンスオリゴヌクレオチドは、肝臓のＡＣＳＬ１の発現を抑制することによって体重の増加の抑制及び血糖値を降下させるため、肝臓での働きにより肝臓毒性を示さないことは、創薬上非常に有用である。 Example 9: Liver toxicity evaluation at the time of single administration of antisense oligo The toxicity in mouse liver was evaluated for the antisense oligonucleotide of the present invention. Specifically, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) contained in plasma were measured as liver toxicity markers. In C57BL / 6J (male 10 weeks old, Claire Japan), about 0.2 mL of antisense oligo solution dissolved in physiological saline (Otsuka raw food injection, Otsuka Pharmaceutical Factory), the dose per mouse is 10 mg / kg, About 0.5 mL of whole blood was collected under somnopentyl anesthesia 3 days, 7 days, and 14 days after administration, and heparin (manufactured by Ajinomoto Pharmaceutical Co., Inc.) was immediately added at a final concentration of 0. It added so that it might become 01U / microliter. AST and ALT in the obtained plasma were measured using a transaminase C2 kit Wako (manufactured by Wako Pure Chemical Industries, Ltd.) according to the attached manual.
The results are shown in Tables 57 to 59. As a result, no significant increase in AST and ALT was observed in the blood of mice administered with the antisense oligonucleotide of the present invention as compared to the physiological saline-administered group, and the antisense oligonucleotide of the present invention showed liver toxicity. It was confirmed that there was no. The antisense oligonucleotide of the present invention suppresses the increase in body weight and suppresses blood glucose level by suppressing the expression of ACSL1 in the liver. Therefore, it does not exhibit liver toxicity due to its action in the liver. Useful.
（試験１：ＡＳＴ）
Figure JPOXMLDOC01-appb-T000066
（試験１：ＡＬＴ）
Figure JPOXMLDOC01-appb-T000067
 (Test 1: AST)
Figure JPOXMLDOC01-appb-T000066
(Test 1: ALT)
Figure JPOXMLDOC01-appb-T000067
（試験２：２０ｍｇ／ｋｇ投与　７日後）
Figure JPOXMLDOC01-appb-T000068
 (Test 2: 7 days after administration of 20 mg / kg)
Figure JPOXMLDOC01-appb-T000068
実施例１０：アンチセンスオリゴヒト配列のｉｎ　ｖｉｖｏ活性評価
　本発明のアンチセンスオリゴヌクレオチドの中で、カニクイザルと１００％一致の配列に関しては、カニクイザルに投与し、肝臓におけるＡＣＳＬ１の発現量を解析することでヒト配列のｉｎ　ｖｉｖｏ活性が評価できる。カニクイザルへ、生理食塩水に溶解したヒト配列のアンチセンスオリゴ溶液を反復投与後、肝臓を摘出し、ｍＲＮＡとタンパク質量の発現変化について調べる。 Example 10: Evaluation of in vivo activity of antisense oligo-human sequences Among the antisense oligonucleotides of the present invention, sequences that are 100% identical to cynomolgus monkeys are administered to cynomolgus monkeys and analyzed for the expression level of ACSL1 in the liver Can evaluate the in vivo activity of human sequences. To human cynomolgus monkeys, a human sequence antisense oligo solution dissolved in physiological saline is repeatedly administered, and then the liver is excised and examined for changes in mRNA and protein expression.
　以上の実施例から明らかなように、本発明アンチセンスオリゴヌクレオチドはＡＣＳＬ１発現抑制活性を示す。従って、本発明化合物は肥満症、肥満関連疾患、糖尿病（特に、ＩＩ型糖尿病）、シンドロームＸ、心臓血管障害又は癌（乳癌、結腸癌、大腸癌、卵巣癌、肺癌等）の等の予防又は治療のための医薬（体重管理用医薬も含む）として非常に有用である。 As is clear from the above examples, the antisense oligonucleotide of the present invention exhibits ACSL1 expression inhibitory activity. Therefore, the compound of the present invention can prevent or prevent obesity, obesity-related diseases, diabetes (particularly type II diabetes), syndrome X, cardiovascular disorder or cancer (breast cancer, colon cancer, colon cancer, ovarian cancer, lung cancer, etc.) or the like. It is very useful as a medicine for treatment (including a medicine for weight management).

Claims (18)

  1. ストリンジェントな条件で、配列番号１の８３２位～８５３位、３６３５位～３６５４位又は３６５９位～３６７６位からなる配列にハイブリダイズ可能な配列を含む、アンチセンスオリゴヌクレオチド。 An antisense oligonucleotide comprising a sequence that can hybridize to the sequence consisting of positions 832 to 853, 3635 to 3654, or 3659 to 3676 of SEQ ID NO: 1 under stringent conditions.
  2. ＡＣＳＬ１の発現を抑制する、請求項１記載のアンチセンスオリゴヌクレオチド。 The antisense oligonucleotide according to claim 1, which suppresses the expression of ACSL1.
  3. 長さが１３～１９塩基である、請求項１又は２記載のアンチセンスオリゴヌクレオチド。 The antisense oligonucleotide according to claim 1 or 2, which has a length of 13 to 19 bases.
  4. ４’位と２’位との間に架橋構造を有する糖修飾ヌクレオシドを１以上含有する、請求項１～３いずれかに記載のアンチセンスオリゴヌクレオチド。 The antisense oligonucleotide according to any one of claims 1 to 3, comprising at least one sugar-modified nucleoside having a cross-linked structure between the 4'-position and the 2'-position.
  5. 該架橋構造が、４’－ＣＨ２－Ｏ－２’又は４’－ＣＯ－ＮＲ１－２’（Ｒ１は、水素原子又はアルキルである）である、請求項４記載のアンチセンスオリゴヌクレオチド。 The antisense oligonucleotide according to claim 4, wherein the bridge structure is 4'-CH 2 -O-2 'or 4'-CO-NR 1 -2' (R 1 is a hydrogen atom or alkyl). .
  6. ヌクレオシド間の１若しくは複数の結合がホスホロチオエート結合である、請求項１～５いずれかに記載のアンチセンスオリゴヌクレオチド。 The antisense oligonucleotide according to any one of claims 1 to 5, wherein one or more bonds between nucleosides are phosphorothioate bonds.
  7. 配列番号３１０～３２０のいずれかに記載の配列、又は、
    配列番号３１０～３２０のいずれかに記載の配列において、１若しくは数個の塩基が欠失、置換若しくは挿入された配列
    を含む、請求項１～６いずれかに記載のアンチセンスオリゴヌクレオチド。     The sequence set forth in any of SEQ ID NOS: 310-320, or
    The antisense oligonucleotide according to any one of claims 1 to 6, comprising a sequence in which one or several bases are deleted, substituted or inserted in the sequence shown in any of SEQ ID NOS: 310 to 320.
  8. 配列番号３１０～３２０のいずれかに記載の配列、又は、
    配列番号３１０～３２０のいずれかに記載の配列において、１若しくは数個の塩基が欠失、置換若しくは挿入された配列
    からなる、請求項７記載のアンチセンスオリゴヌクレオチド。     The sequence set forth in any of SEQ ID NOS: 310-320, or
    The antisense oligonucleotide according to claim 7, wherein the antisense oligonucleotide comprises a sequence in which one or several bases are deleted, substituted or inserted in the sequence shown in any of SEQ ID NOs: 310 to 320.
  9. 配列番号３６３、３６５～３６９、３７５～３７８、３８０～３８９、３９２～３９４、３９８、４００～４０３、４０５、４０８～４１３、４１５～４１８、４２３～４２５、４２８、４３０～４３３、４３６、４３７、４４０、４４３～４４７、４５０、４５２、４５３、４５６～４５８、４６２、４６４～４６９、４７３～４７５、４７７、４７８、４８０～４８２、４８４～４８６、４８９、４９１～４９８、５００若しくは５０２のいずれかに記載の配列、又は、
    配列番号３６３、３６５～３６９、３７５～３７８、３８０～３８９、３９２～３９４、３９８、４００～４０３、４０５、４０８～４１３、４１５～４１８、４２３～４２５、４２８、４３０～４３３、４３６、４３７、４４０、４４３～４４７、４５０、４５２、４５３、４５６～４５８、４６２、４６４～４６９、４７３～４７５、４７７、４７８、４８０～４８２、４８４～４８６、４８９、４９１～４９８、５００若しくは５０２のいずれかに記載の配列において、１若しくは数個の塩基が欠失、置換若しくは挿入された配列
    を含む、アンチセンスオリゴヌクレオチド。     SEQ ID NOs: 363, 365-369, 375-378, 380-389, 392-394, 398, 400-403, 405, 408-413, 415-418, 423-425, 428, 430-433, 436, 437, 440, 443-447, 450, 452, 453, 456-458, 462, 464-469, 473-475, 477, 478, 480-482, 484-486, 489, 491-498, 500 or 502 Or the sequence described in
    SEQ ID NOs: 363, 365-369, 375-378, 380-389, 392-394, 398, 400-403, 405, 408-413, 415-418, 423-425, 428, 430-433, 436, 437, 440, 443-447, 450, 452, 453, 456-458, 462, 464-469, 473-475, 477, 478, 480-482, 484-486, 489, 491-498, 500 or 502 An antisense oligonucleotide comprising a sequence wherein one or several bases are deleted, substituted or inserted in the sequence described in 1.
  10. ＡＣＳＬ１の発現を抑制する、請求項９記載のアンチセンスオリゴヌクレオチド。 The antisense oligonucleotide according to claim 9, which suppresses the expression of ACSL1.
  11. 長さが１３～１９塩基である、請求項９又は１０記載のアンチセンスオリゴヌクレオチド。 The antisense oligonucleotide according to claim 9 or 10, which has a length of 13 to 19 bases.
  12. ３６３、３６５～３６９、３７５～３７８、３８０～３８９、３９２～３９４、３９８、４００～４０３、４０５、４０８～４１３、４１５～４１８、４２３～４２５、４２８、４３０～４３３、４３６、４３７、４４０、４４３～４４７、４５０、４５２、４５３、４５６～４５８、４６２、４６４～４６９、４７３～４７５、４７７、４７８、４８０～４８２、４８４～４８６、４８９、４９１～４９８、５００若しくは５０２のいずれかに記載の配列、又は、
    ３６３、３６５～３６９、３７５～３７８、３８０～３８９、３９２～３９４、３９８、４００～４０３、４０５、４０８～４１３、４１５～４１８、４２３～４２５、４２８、４３０～４３３、４３６、４３７、４４０、４４３～４４７、４５０、４５２、４５３、４５６～４５８、４６２、４６４～４６９、４７３～４７５、４７７、４７８、４８０～４８２、４８４～４８６、４８９、４９１～４９８、５００若しくは５０２のいずれかに記載の配列において、１若しくは数個の塩基が欠失、置換若しくは挿入された配列
    からなる、請求項９～１１いずれかに記載のアンチセンスオリゴヌクレオチド。     363, 365-369, 375-378, 380-389, 392-394, 398, 400-403, 405, 408-413, 415-418, 423-425, 428, 430-433, 436, 437, 440, 443 to 447, 450, 452, 453, 456 to 458, 462, 464 to 469, 473 to 475, 477, 478, 480 to 482, 484 to 486, 489, 491 to 498, 500 or 502 An array of
    363, 365-369, 375-378, 380-389, 392-394, 398, 400-403, 405, 408-413, 415-418, 423-425, 428, 430-433, 436, 437, 440, 443 to 447, 450, 452, 453, 456 to 458, 462, 464 to 469, 473 to 475, 477, 478, 480 to 482, 484 to 486, 489, 491 to 498, 500 or 502 The antisense oligonucleotide according to any one of claims 9 to 11, comprising a sequence in which one or several bases are deleted, substituted or inserted.
  13. ４’位と２’位との間に架橋構造を有する糖修飾ヌクレオシドを１以上含有する、請求項９～１２いずれかに記載のアンチセンスオリゴヌクレオチド。 The antisense oligonucleotide according to any one of claims 9 to 12, comprising at least one sugar-modified nucleoside having a cross-linked structure between the 4'-position and the 2'-position.
  14. 該架橋構造が、４’－ＣＨ２－Ｏ－２’又は４’－ＣＯ－ＮＲ１－２’（Ｒ１は、水素原子又はアルキルである）である、請求項１３記載のアンチセンスオリゴヌクレオチド。 The antisense oligonucleotide according to claim 13, wherein the bridge structure is 4'-CH 2 -O-2 'or 4'-CO-NR 1 -2' (R 1 is a hydrogen atom or alkyl). .
  15. ヌクレオシド間の１若しくは複数の結合がホスホロチオエート結合である、請求項９～１４いずれかに記載のアンチセンスオリゴヌクレオチド。 The antisense oligonucleotide according to any one of claims 9 to 14, wherein the one or more bonds between nucleosides are phosphorothioate bonds.
  16. 請求項１～１５いずれかに記載のアンチセンスオリゴヌクレオチドを含有する医薬組成物。 A pharmaceutical composition comprising the antisense oligonucleotide according to any one of claims 1 to 15.
  17. ＡＣＳＬ１が関連する疾患の予防又は治療のために用いる、請求項１６記載の医薬組成物。 The pharmaceutical composition according to claim 16, which is used for prevention or treatment of a disease associated with ACSL1.
  18. 該疾患が、肥満症又はＩＩ型糖尿病である、請求項１７記載の医薬組成物。 The pharmaceutical composition according to claim 17, wherein the disease is obesity or type II diabetes.
PCT/JP2014/071011 2013-08-09 2014-08-08 Antisense oligonucleotide against acsl1 WO2015020194A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006506976A (en) * 2002-08-14 2006-03-02 ファルマシア・コーポレーション Antisense regulation of acyl-CoA synthetase 1 expression
WO2010079819A1 (en) * 2009-01-08 2010-07-15 塩野義製薬株式会社 Pharmaceutical composition for treating obesity or diabetes
WO2011052436A1 (en) * 2009-10-29 2011-05-05 国立大学法人大阪大学 Bridged artificial nucleoside and nucleotide

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006506976A (en) * 2002-08-14 2006-03-02 ファルマシア・コーポレーション Antisense regulation of acyl-CoA synthetase 1 expression
WO2010079819A1 (en) * 2009-01-08 2010-07-15 塩野義製薬株式会社 Pharmaceutical composition for treating obesity or diabetes
WO2011052436A1 (en) * 2009-10-29 2011-05-05 国立大学法人大阪大学 Bridged artificial nucleoside and nucleotide

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