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WO2015020194A1 - Oligonucléotide antisens contre acsl1 - Google Patents

Oligonucléotide antisens contre acsl1 Download PDF

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Publication number
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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Prior art keywords
antisense oligonucleotide
sequence
seq
acsl1
substituted
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Japanese (ja)
Inventor
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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Shionogi and Co Ltd
University of Osaka NUC
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Osaka University NUC
Shionogi and Co Ltd
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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

　Il a été constaté qu'un oligonucléotide antisens comprenant une séquence qui peut s'hybrider avec des séquences comprenant les positions 832-853, 3635-3654 ou 3659-3676 de SEQ ID No. 1 présente une plus forte activité inhibitrice sur l'expression de ACSL1. Cette composition pharmaceutique contient l'oligonucléotide antisens contre ACSL1 en tant que principe actif de celui-ci, et est utile pour la prévention et le traitement de l'obésité et du diabète de type II.
PCT/JP2014/071011 2013-08-09 2014-08-08 Oligonucléotide antisens contre acsl1 Ceased WO2015020194A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118638786A (zh) * 2024-06-12 2024-09-13 云南大学 靶向acsl4基因的反义寡核苷酸及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006506976A (ja) * 2002-08-14 2006-03-02 ファルマシア・コーポレーション アシル−ＣｏＡシンテターゼ１発現のアンチセンス調節
WO2010079819A1 (fr) * 2009-01-08 2010-07-15 塩野義製薬株式会社 Composition pharmaceutique destinée au traitement de l'obésité ou du diabète
WO2011052436A1 (fr) * 2009-10-29 2011-05-05 国立大学法人大阪大学 Nucléoside et nucléotide artificiels pontés

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006506976A (ja) * 2002-08-14 2006-03-02 ファルマシア・コーポレーション アシル−ＣｏＡシンテターゼ１発現のアンチセンス調節
WO2010079819A1 (fr) * 2009-01-08 2010-07-15 塩野義製薬株式会社 Composition pharmaceutique destinée au traitement de l'obésité ou du diabète
WO2011052436A1 (fr) * 2009-10-29 2011-05-05 国立大学法人大阪大学 Nucléoside et nucléotide artificiels pontés

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118638786A (zh) * 2024-06-12 2024-09-13 云南大学 靶向acsl4基因的反义寡核苷酸及其应用
CN118638786B (zh) * 2024-06-12 2025-01-21 云南大学 靶向acsl4基因的反义寡核苷酸及其应用

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