TW201336860A - Antisense oligonucleotide for acsl1 - Google Patents

Abstract

It is found that an antisense oligonucleotide comprising a sequence complementary to a sequence lying between position-95 and position-109, position-176 and position-192, position-467 and position-484, position-940 and position-954, position-1017 and position-1032, position-1102 and position-1116, position-1176 and position-1197, position-1222 and position-1236, position-1727 and position-1743, position-1858 and position-1873, position-1946 and position-1960, position-2294 and position-2308, position-2360 and position-2377, position-2449 and position-2469, position-2605 and position-2624, position-2689 and position-2703, position-2950 and position-2964, position-3424 and position-3438 or position-3591 and position-3605 in the sequence represented by SEQ ID NO: 2 has an excellent inhibitory activity on the expression of ACSL1. A pharmaceutical composition containing the antisense oligonucleotide for ACSL1 according to the present invention as an active ingredient is useful for the prevention or treatment of obesity or type-II diabetes.

Description

Antisense oligonucleotide against ACSL1 Field of invention

The present invention relates to a counter-oligonucleotide directed against ACSL1 (acyl-CoA synthetase long-chain family member 1). In more detail, it is a useful anti-intentional oligonucleus for ACSL1 as a preventive or therapeutic drug for obesity or diabetes (especially type II diabetes) (also including a pharmaceutical composition for weight management). Glycosylate.

Background of the invention

Obesity refers to a state in which adipose tissue is systemically increased, and occurs when the amount of heat taken during a long period of time is greater than the amount of calories consumed. Obesity can be classified into visceral fat obesity and subcutaneous fat obesity. Visceral fat type obesity is an obesity in which the accumulation of fat in the peritoneal cavity around the mesenteric and mesenteric is increased, and is considered to cause diabetes (especially type II diabetes with insulin resistance), arteriosclerosis, liver One of the culprit of illness, heart disease, etc. has become a big problem in modern society.

Diabetes is a disease associated with persistent hyperglycemia and is thought to be produced by most environmental factors and genetic factors. The main regulator of blood glucose in the body is insulin, and the high blood sugar is due to insulin deficiency, or Caused by excess factors that impede its effects (eg, genetic factors, lack of exercise, obesity, stress, etc.). There are two main types of diabetes, which are classified as: Type I diabetes, which is caused by low insulin secretion of pancreas caused by autoimmune diseases; and Type II diabetes is caused by Sustained high insulin secretion caused by pancreatic failure caused by pancreatic insulin secretion. More than 95% of Japanese diabetic patients are type 2 diabetes. Today, the increase in the number of patients with lifestyle changes has become a problem.

An aldehyde (hereinafter, abbreviated as ACS) belonging to the acetyl group-CoA synthetase family is an enzyme that converts from a long-chain fatty acid to an ethyl amide-based CoA. Since the acetamidine-based CoA system acts as a matrix in intracellular lipid synthesis and fatty acid decomposition or elongation, the ACS plays a central role in intracellular lipid metabolism and even in intracellular signaling using lipids. Further, ACS also has an intake of fatty fats (see Non-Patent Document 1).

ACS has heretofore been identified as five isozymes (ACS1, 3, 4, 5, and 6) in which matrix selectivity and intracellular localization are different (see Non-Patent Document 2). It is generally known that ACSL1 (GenBank: NM_001995), which is one of the family's enzymes, is mainly expressed in liver and adipose tissue, and catalyzes the synthesis of triglyceride (TG). Furthermore, the ACSL4 and ACSL5 lines are mainly expressed in the liver, while the ACSL3 and ACSL6 are mainly expressed in the brain.

TriacsinC is known as an ACS inhibitor, and it is reported that this compound can block 1, 3, and 4 of the five isozymes (see Non-Patent Document 3). Regarding this compound, it has been reported that it inhibits TG accumulation in HuH7 cells belonging to human liver cancer cell lines (refer to Non-Patent Document 4) or in human beings. In CCD cells of normal skin fibroblasts, synthesis of bis-mercaptoglycerol, cholesterol ester, and phospholipid is inhibited (see Non-Patent Document 5).

There are reports on the relationship between ACSL1 and various cancers (for example, refer to Patent Documents 1 to 3), and there are reports that ACSL1 will become cirrhosis and liver fibrosis (refer to Patent Document 4) and bronchial asthma (see patent). Biomarkers of document 5).

Further, in Patent Document 6, it is taught that siRNA is used to suppress the expression of ACSL1 in the liver, thereby suppressing the increase in body weight and lowering the blood sugar level, and the ACSL1 expression inhibitor can be used for the treatment of obesity or type II diabetes or It is prevention.

In Patent Document 7, an anti-intentional compound targeting ACSL1 is disclosed. In the specification, although the antisense oligonucleotide of 3615 is described, only the predicted value regarding the affinity with the target field is described, but the data on the inhibition of the expression of ACSL1 is not described.

Advanced technical literature Patent literature

[Patent Document 1] International Publication No. 07/010628

[Patent Document 2] International Publication No. 07/117038

[Patent Document 3] International Publication No. 03/3004989

[Patent Document 4] Japanese Patent Laid-Open No. 2007-252366

[Patent Document 5] Japanese Patent Laid-Open No. 2004-121218

[Patent Document 6] International Publication No. 2010/079819

[Patent Document 7] International Publication No. 04/016749

Non-patent literature

[Non-Patent Document 1] Biocheical. J, Vol. 323, pp. 1-12, 1997

[Non-Patent Document 2] J. Nutr, Vol. 132, pp. 2123-2126, 2004

[Non-Patent Document 3] Biochemistry, Vol. 44, pp. 1635-1642, 2005

[Non-Patent Document 4] J. Lipid. Res, Vol. 48, pp. 1280-1292, 2007

[Non-Patent Document 5] Biochem. J, Vol. 324, pp. 529-534, 1997

Summary of invention

It is an object of the present invention to provide a novel anti-initiative oligonucleotide which has excellent ACSL1 expression inhibitory activity.

As a result of intensive studies, the present inventors succeeded in synthesizing a novel antisense oligonucleotide having excellent ACSL1 expression inhibitory activity (knockdown activity). Further, the present inventors have found a target field in the mRNA of ACSL1, particularly related to the knockdown activity of the reverse oligonucleotide. In Patent Document 7, although the antisense oligonucleotide against ACSL1 of 3615 is described, only the predicted value regarding the affinity with the target field is described, and the information on the inhibition of the expression of ACSL1 is not described. It is technically common to have high affinity reversal and not necessarily have high target gene expression inhibition (Antisense Drug Technology Principles, Strategies, and Applications, CRC Press; 2nd edition, 2007, page 120-122, Figure 5.3a). Therefore, from the description of Patent Document 7, it is impossible to predict ACSL1. The field of mRNA can be used as the subject of antisense oligonucleotides. The anti-intentional oligonucleotide of the present invention binds to a specific target field discovered by the present inventors and the like, and exhibits excellent ACSL1 expression inhibitory activity.

Further, the present inventors have found that the anti-intelligence oligonucleotide of the present invention does not exhibit an inhibitory effect on other isozymes (ACSL3 and ACSL5), but has an inhibitory effect on the expression of ACSL1. Such anti-intentional oligonucleotides with high specificity to the target sequence are useful in medicine.

Further, the anti-intentional oligonucleotide of the present invention is very safe for use in medicine because of its metabolic stability, water solubility, and low toxicity.

That is, the present invention relates to the following.

(1) A reverse oligonucleotide comprising a sequence which can be heterozygous under stringent conditions to a sequence consisting of: 95 to 109, 176 to SEQ ID NO: 1. 192, 467 to 484, 940 to 954, 1017 to 1032, 1102 to 1116, 1176 to 1197, 1222 to 1236, 1727 to 1743, 1858 to 1873 1,946 to 1960, 2294 to 2308, 2360 to 2377, 2449 to 2469, 2605 to 2624, 2689 to 2703, 2950 to 2964, 3424 to 3438 or 3591 to 3605.

(2) A counter-oligonucleotide as in (1) which inhibits the expression of ACSL1.

(3) A reverse oligonucleotide of (1) or (2), which is 13 to 19 bases in length.

(4) The anti-intelligence oligonucleotide according to any one of (1) to (3) which contains one or more glycosyl-modified nucleosides having a bridging structure at the 4'-position and the 2'-position.

(5) The anti-intentional oligonucleotide of (4), the bridging structure is -CH2-O- or -CO-NR1-(R1 is a hydrogen atom or an alkyl group).

(6) The anti-intentional oligonucleotide according to any one of (1) to (5), which comprises SEQ ID NO: 6 to 11, 13 to 18, 20 to 22, 24 to 30, 32, 35 to 37, Sequence of 46, 48, 49, 52 to 54, 57 to 59, 64 to 68 or 70; or, contained in SEQ ID NO: 6 to 11, 13 to 18, 20 to 22, 24 to 30, 32, 35 to 37 In the sequence of 46, 48, 49, 52 to 54, 57 to 59, 64 to 68 or 70, there are sequences in which one or several bases are deleted, replaced or inserted.

(7) A counter-oligonucleotide as in (6), which consists of the following sequences: Nos. 6 to 11, 13 to 18, 20 to 22, 24 to 30, 32, 35 to 37, 46, 48, Sequence of 49, 52 to 54, 57 to 59, 64 to 68 or 70; or in sequence numbers 6 to 11, 13 to 18, 20 to 22, 24 to 30, 32, 35 to 37, 46, 48, 49 In the sequence of 52 to 54, 57 to 59, 64 to 68 or 70, there are 1 or several bases deleted, substituted, inserted or added.

(8) A pharmaceutical composition comprising the anti-intelligence oligonucleotide according to any one of (1) to (7).

(9) The pharmaceutical composition according to (8), which is for use in the prevention or treatment of an ACSL1-related disease.

(10) The pharmaceutical composition according to (9), wherein the disease is obesity or type II diabetes.

(11) A method for the prophylaxis or treatment of an ACSL1-related disease, which comprises the reverse oligonucleotide of any one of (1) to (7).

(12) The method of prevention or treatment according to (11), wherein the disease is obesity or Type II diabetes.

(13) The use of a reverse oligonucleotide of any one of (1) to (7) for the prevention of an ACSL1-related disease or for the manufacture of a therapeutic agent.

(14) The use of the reverse oligonucleotide of (13), wherein the disease is obesity or type II diabetes.

(15) The anti-intelligence oligonucleotide according to any one of (1) to (7), which is for use in the prevention or treatment of an ACSL1-related disease.

(16) The anti-intentional oligonucleotide of (15), wherein the disease is obesity or type II diabetes.

The anti-intentional oligonucleotide of the present invention exhibits excellent ACSL1 expression inhibitory activity, and is useful as a pharmaceutical, particularly for an ACSL1-related disease, for example, obesity, obesity-related diseases, diabetes (especially type II diabetes), X Symptoms, palpitations, or cancer (breast cancer, colon cancer, colon cancer, ovarian cancer, lung cancer, etc.) are also very useful for the prevention or treatment of drugs (including medicine for weight management).

Fig. 1 shows the knockdown efficiency of the antisense oligonucleotide (5 nM and 20 nM) of the antisense oligonucleotide of the present invention which has been introduced into cells with Lipofectamine LTX reagent in HepG2 cells.

[Fig. 2] The knockdown effect of the antisense oligonucleotide of the present invention on the ACSL1 protein in HepG2 cells

[Fig. 3] The anti-intentional oligonucleotide pair of the present invention is in HepG2 cells. Cross-evaluation results of ACSL3 and ACSL5

Fig. 4 shows the knockdown effect on the ACSL1 protein when a single anti-infectious oligonucleotide (AON No. 197 and 203) of the present invention is administered in vivo (in vivo).

Fig. 5 shows the knockdown effect on the ACSL1 protein when a single anti-infectious oligonucleotide (AON No. 196 and 203) of the present invention is administered in vivo (in vivo).

Fig. 6 shows the knockdown effect on the ACSL1 protein when the anti-intent oligonucleotide (AON No. 203) of the present invention is administered in vivo (in vivo).

[Fig. 7] Weight change when the anti-intent oligonucleotide (AON No. 203) of the present invention is administered in vivo (in vivo)

Form for implementing the invention

The terms used in the present specification are used in the ordinary sense of the art, unless otherwise specified.

In the present invention, genetic manipulation methods well known in the art can be used. For example, a method such as that described in Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001), Current Protocols in Molecular Biology, John Wiley & Sons (2003), and the like can be mentioned.

The invention will be described in detail below.

"Analytic oligonucleotide" (AON) refers to an oligonucleotide complementary to an mRNA, an mRNA precursor or an ncRNA of a target gene; A single strand of DNA, RNA, and/or a similar analog thereof. The antisense oligonucleotide forms a double strand with the target mRNA, mRNA precursor or ncRNA, thereby suppressing the function of mRNA, mRNA precursor or ncRNA. As far as the "anti-intelligence oligonucleotide" is concerned, it means not only the mRNA, the mRNA precursor or the ncRNA which are the target, but can be hybridized with the mRNA, the mRNA precursor or the ncRNA under stringent conditions. The existence of one or several mismatches is also included.

Oligonucleotide refers to a combination of a plurality of identical or different nucleosides. "Nucleoside" refers to a compound in which a nucleobase is bonded to a sugar by an N-glycoside. "Nucleotide" refers to a compound in which a phosphate group is bonded to a sugar of a nucleoside.

A similar system of DNA or RNA refers to a molecule having a structure similar to DNA or RNA. For example, peptide nucleic acid (PNA) or the like can be mentioned.

ncRNA (non-coding RNA) is a generic term for RNA that does not translate into protein and organic energy. For example, rRNA, tRNA, miRNA, etc. are mentioned.

1 or a few mismatches refer to 1 to 5, preferably 1 to 3, more preferably 1 or 2 mismatches.

The target gene of the anti-intention oligonucleotide of the present invention may, for example, be ACSL1. For example, human ACSL1, murine ACSL1, and the like can be mentioned, but are not limited thereto.

"ACSL1" is a well-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 the mouse ACSL1 (GenBank: NM_007981) is recorded in the sequence of the sequence listing. No. 3, the amino acid sequence is described in SEQ ID NO: 4. The "ACSL1" in the present invention is not limited to those sequences, but means that if the function of the protein of SEQ ID NO: 2 or 4 is maintained, the number of variants or variants of the amino acid or DNA is not limited.

The inverse oligonucleotide of the present invention is 6 to 50 bases in length. 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 Base, 18 bases, 19 bases.

As the "anti-intelligence oligonucleotide" of the present invention, for example, (a) a reverse oligonucleotide comprising a sequence which can be hybridized under stringent conditions to the sequence of SEQ ID NO: 1 from position 95 to position 109, position 176 to position 192, position 467 to position 484, 940 to 954, 1017 to 1032, 1102 to 1116, 1176 to 1197, 1222 to 1236, 1727 to 1743, 1858 to 1873, 1946 to 1960, 2294 Up to 2308, 2360 to 2377, 2449 to 2469, 2605 to 2624, 2689 to 2703, 2950 to 2964, 3424 to 3438, or 3591 to 3605; or Yes (b) a reverse oligonucleotide comprising a sequence which can be hybridized under stringent conditions to the sequence of 183 to 197, 219 to 235, and 442 to 456 of the sequence of SEQ ID NO:3, 513 to 530, 593 to 607, 641 to 655, 919 to 933, 941 to 955, 959 to 973, 978 to 993, 998 to 1013, 1222 To 1243, 1268 to 1282, 1625 to 1639, 1993 to 2007, 2066 to 2081, 2409 to 2423, 2497 to 2525, 2651 to 2670 Bits, 2685 to 2700, 2738 to 2756, 2764 to 2788, 3331 to 3347, 3503 to 3517, or 3663 to 3684.

The subject areas of (a) are the domains of human ACSL1 mRNA, particularly those associated with the knockdown activity of anti-intelligent oligonucleotides. The subject areas of (b) are the areas of the mRNA of murine ACSL1, particularly those associated with the knockdown activity of anti-intelligent oligonucleotides. Under stringent conditions, as long as it is a sequence which can be hybridized to the target field, it is included in the anti-intentional oligonucleotide of the present invention regardless of the length or the presence or absence of a nucleotide modification.

The ACSL1 expression inhibitory activity (knockdown activity) can be measured by a known method. For example, it can be measured by the method described in Example 5 (1), (3), Example 6, or Example 7 which will be described later.

As the anti-intelligence oligonucleotide of the present invention, preferred is: (c) an inverted oligonucleotide comprising a sequence which can be hybridized under stringent conditions to the sequence of 176th position of the sequence of SEQ ID NO: 1. Sequences of 192, 467 to 484, 1176 to 1197, 2449 to 2469, or 2605 to 2624; or (d) comprise hybridization to the following sequences under stringent conditions Sequence antisense oligonucleotide: Sequence number 3 sequence 219 to 235, 513 to 530, 1222 to 1243, 2497 to 2525, 2651 to 2670, 2685 to 2700 , 2738 to 2756, 2764 to 2788 or 3663 to 3684.

Specific examples of the anti-intelligence oligonucleotide of the present invention include at least 70% or more, preferably 80% or more, of a complementary sequence to the following sequence. Preferably, it is 90% or more, and most preferably 95% or more of the same antisense oligonucleotide. Here, the identity is expressed in terms of a score by, for example, a search program BLAST using an algorithm developed by Altschul et al. (The Journal of Molecular Biology, 215, 403-410 (1990).).

(a) Sequences of sequence number 1 from 95 to 109, 176 to 192, 467 to 484, 940 to 954, 1017 to 1032, 1102 to 1116, and 1176 to 1197 1,222 to 1236, 1727 to 1743, 1858 to 1873, 1946 to 1960, 2294 to 2308, 2360 to 2377, 2449 to 2469, 2605 to 2624, 2689 a sequence of 2703, 2950 to 2964, 3424 to 3438, or 3591 to 3605; or (b) Sequence 183 to 197 to 197, 219 to 235, 442 To 456, 513 to 530, 593 to 607, 641 to 655, 919 to 933, 941 to 955, 959 to 973, 978 to 993, 998 to 1013, 1222 to 1243, 1268 to 1282, 1625 to 1639, 1993 to 2007, 2066 to 2081, 2409 to 2423, 2497 to 2525, 2651 to 2670 2685 to 2700, 2738 to 2756, 2764 to 2788, 3331 to 3347, 3503 to 3517 or 3663 to 3684.

"Stringent conditions" means that only the opposite oligonucleotide forms a hybrid with the following sequence (so-called specific hybridization), and the base sequence which does not have the same function does not form a hybrid with the specific sequence (so-called non- Conditions for specific heterozygous): (a) Sequence number 1 sequence from 95 to 109, 176 to 192, 467 to 484, 940 to 954, 1017 to 1032, and 1102 To 1116, 1176 to 1197, 1222 to 1236, 1727 to 1743, 1858 to 1873, 1946 to 1960, 2294 to 2308, 2360 to 2377, 2449 to 2469 Bit, sequence from 2605 to 2624, 2689 to 2703, 2950 to 2964, 3424 to 3438, or 3591 to 3605, or (b) sequence 183 to 197 to 197 Position, 219 to 235, 442 to 456, 513 to 530, 593 to 607, 641 to 655, 919 to 933, 941 to 955, 959 to 973, 978 to 993, 998 to 1013, 1222 to 1243, 1268 to 1282, 1625 to 1639, 1993 to 2007, 2066 to 2081, 2409 to 2423, 2497 Up to 2525, 2651 to 2670, 2685 to 2700, 2738 to 2756, 2764 to 2788, 3331 to 3347, 3503 to 3517 or 3663 to 3684.

Those skilled in the art can easily select such conditions by changing the temperature of the hybrid reaction and washing, the salt concentration of the hybrid reaction solution and the cleaning solution, and the like. Specifically, the following conditions are listed as an example of the stringent conditions of the present invention, but are not limited thereto: 6 x SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6 x SSPE (3 M NaCl). , 0,2M NaH ₂ PO ₄ , 20 mM EDTA. 2Na, pH 7.4) and mixed at 42 ° C, and then washed under conditions of 0.5 × SSC at 42 ° C. As the hybrid method, a technique conventionally known in the art, for example, Southern dot impurity method or the like can be used. Specifically, 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 can be followed. It is carried out by the method described in Second Edition (1995) (Oxford University Press).

As the anti-intelligence oligonucleotide of the present invention, for example, a reverse oligonucleotide comprising the following sequences: SEQ ID Nos. 6 to 11, 13 to 18, 20 to 22, 24 to 30, 32, 35 to 37 a sequence of 46, 48, 49, 52 to 54, 57 to 59, 64 to 68 or 70; or in sequence numbers 6 to 11, 13 to 18, 20 to 22, 24 to 30, 32, 35 to 37, A sequence in which a sequence of 46, 48, 49, 52 to 54, 57 to 59, 64 to 68 or 70 has one or several bases deleted, replaced or inserted. These lines have human ACSL1 expression inhibitory activity, respectively. As long as the sequence is contained, it is included in the antisense oligonucleotide of the present invention regardless of the length or the presence or absence of the nucleotide modification.

Further, as the anti-intelligence oligonucleotide of the present invention, for example, a reverse oligonucleotide comprising the following sequences: SEQ ID NOs: 6 to 13, 30, 72 to 75, 77 to 80, 82 to 93, 95 a sequence of 100 to 104, 109, 110, 113, 114, 116 to 127, 129, 131 to 141, 143 to 147, 151, 153 to 155 or 156; or in sequence numbers 6 to 13, 30, 72 In the sequence of 75, 77 to 80, 82 to 93, 95, 100 to 104, 109, 110, 113, 114, 116 to 127, 129, 131 to 141, 143 to 147, 151, 153 to 155 or 156 , a sequence with 1 or a few base deletions, substitutions, or insertions.

These lines contain murine ACSL1 expression inhibitory activity, respectively. As long as it contains The sequence, regardless of the length or the presence or absence of a nucleotide modification, is included in the antisense oligonucleotide of the present invention.

Specific examples of the anti-intelligence oligonucleotide of the present invention include reverse nucleotides consisting of the following sequences: SEQ ID NO: 6 to 11, 13 to 18, 20 to 22, 24 to 30, 32 a sequence of 35 to 37, 46, 48, 49, 52 to 54, 57 to 59, 64 to 68 or 70; or in sequence numbers 6 to 11, 13 to 18, 20 to 22, 24 to 30, 32, In the sequence of 35 to 37, 46, 48, 49, 52 to 54, 57 to 59, 64 to 68 or 70, one or several bases are deleted, substituted, inserted or added. More preferably, it is a reverse oligonucleotide consisting of SEQ ID NO: 6 to 11 or 24 to 28; or in the sequence of SEQ ID NO: 6 to 11 or 24 to 28, having 1 or several bases. A sequence of deletions, substitutions, insertions, or additions.

These lines have human ACSL1 expression inhibitory activity, respectively. Any of the nucleotide sequences may be included in the antisense oligonucleotide of the present invention as long as it is composed of the sequence.

Further, as the anti-intelligence oligonucleotide of the present invention, specifically, a reverse oligonucleotide consisting of the following: SEQ ID NOs: 6 to 13, 30, 72 to 75, 77 to 80, 82 a sequence of 93, 95, 100 to 104, 109, 110, 113, 114, 116 to 127, 129, 131 to 141, 143 to 147, 151, 153 to 155 or 156; or in sequence numbers 6 to 11 In the sequence of 13 to 18, 20 to 22, 24 to 30, 32, 35 to 37, 46, 48, 49, 52 to 54, 57 to 59, 64 to 68 or 70, there is 1 Or a sequence of base deletions, substitutions, insertions, or additions.

These lines have murine ACSL1 expression inhibitory activity, respectively. Any of the nucleotide sequences may be included in the antisense oligonucleotide of the present invention as long as it is composed of the sequence.

Further, "1 or several bases" means 1 to 5, preferably 1 to 3, more preferably 1 or 2 bases. Even if a deletion, substitution, insertion or additional modification is used, it is included in the antisense oligonucleotide of the present invention as long as it exhibits an inhibitory effect on the target gene (for example, ACSL1).

The anti-intelligence oligonucleotide of the present invention not only has the ACSL1 expression inhibitory activity, but also has practical utility as a drug, and has any of the following characteristics or all of the excellent characteristics.

a) The blocking effect on CYP enzymes (eg, CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, etc.) is weak.

b) exhibits good pharmacokinetic properties such as high bioavailability, moderate clearance, and the like.

c) High metabolic stability.

d) is not mutagenic.

e) The risk of the cardiovascular system is low.

f) exhibits high solubility.

g) Express ACSL1 specificity.

h) does not show hepatotoxicity.

In the anti-intelligence oligonucleotide of the present invention, the nucleotide may also be modified. The anti-intentional oligonucleotide to which the appropriate modification is applied has any one of the following, or all of the characteristics, as compared with the unmodified anti-intelligence oligonucleotide.

a) High affinity to the target gene.

b) High resistance to nucleases.

c) Improve pharmacokinetic properties.

d) Increased organizational mobility.

Therefore, the modified anti-intelligence oligonucleotide becomes difficult to be decomposed in the living body as compared with the unmodified anti-intelligence oligonucleotide, and can stably hinder the expression of the target gene.

Any of the nucleotide modifications well known in the art can be utilized in the anti-intentional oligonucleotides of the present invention. In terms of modification of a nucleotide, a phosphoric acid modification, a nucleic acid base modification, and a sugar group modification are generally known.

The phosphoric acid modification may, for example, be a phosphodiester bond possessed by a natural nucleic acid, S-oligo (thiophosphate), D-oligo (phosphodiesterate), M-oligo (methylphosphonate), boron. Alkylated phosphates, etc.

Examples of the nucleic acid base modification include 5-methylcytosine, 5-hydroxymethylcytosine, and 5-propynylcytosine.

The glycosyl modification system may, for example, be 2'-O-CH2-CH2-O-CH3 (2'MOE), LNA (Locked nucleic acid), guanamine BNA (Bridged nucleic acid, AmNA) or the like.

Methods for modifying and modifying nucleotides well known in the art are also disclosed, for example, in 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. 2003/068795, International Publication No. 2011/052436, International Publication No. 2004 /016749, International Publication No. 2005/083124, International Publication 2007/143315, International Publication No. 2009/071680, Japanese Patent Application No. 2013-004735, and the like.

The modifications exemplified in the anti-intentional oligonucleotides of the present invention are specifically exemplified below, but the modifications are not limited to those.

(1) S-oligo(thiophosphoric acid)

The oxygen atom of the phosphate group of the phosphodiester bond between the nucleosides is replaced with a sulfur atom. This modification is incorporated into the oligonucleotide according to a known method. A reverse oligonucleotide having 1 or a plurality of such modifications in an oligonucleotide is referred to as an S-oligo (thiophosphate type).

(2) LNA (Locked nucleic acid)

The bicyclic sugar moiety is formed by a 2 ' -hydroxy group bonded to the 4 ' carbon atom of the sugar ring of the nucleotide through a suitable bridge. A preferred linkage is a methylene (-CH2-) group bridging a 2 ' oxygen atom with a 4 ' carbon atom. Specific examples of the LNA and the preparation method thereof are described in International Publication No. 98/39352, International Publication No. 2003/068795, International Publication No. 2005/021570, and the like.

(3) BNA (Bridged nucleic acid, AmNA)

A bicyclic sugar moiety can be formed by forming a guanamine bond between the 2 ' amine group of the sugar ring of the nucleotide and the carbonyl group extending from 4 ' . Specific examples of the indoleamine BNA and a method for preparing the same are described in International Publication No. 2011/052436.

(4) Nucleotide sugar 2 ' position modification

The OH at the 2 ' position of the sugar of the nucleotide is replaced with an -O-alkyl group (e.g., -O-methyl, -O-ethyl, -O-ethoxy, etc.) or -F. This modification is incorporated into the oligonucleotide according to a known method.

The reverse oligonucleotide of the present invention is preferably a gapmer. Gapmer refers to the field containing the central field ("gap") and the sides of the central field, the wings ("5 ' wings on the 5 ' side" or the "3 ' wings" on the 3 ' side), and each The wing contains at least one oligonucleotide that modifies the nucleotide. The modification of the modified nucleotide may be any one of a phosphoric acid modification, a base modification, and a sugar group modification. The type, number, and position of the modifications in one wing may be the same as or different from the type, number, and position of the modification of the other wing.

Are preferred, the present invention based antisense "containing one or more glycosylation structure having a bridge between the 4 'position and 2' position of the modified nucleoside." Preferably, it is a gapmer. In the "5 ' wing" and/or the "3 ' wing", there are more than one glycosyl-modified nucleoside having a bridging structure between the 4 ' position and the 2 ' position, preferably 1 to 5, More preferably, it contains 2 to 3. Further, it may further contain a phosphoric acid modification, a base modification, or another glycosyl modification.

"Glycosyl structure having a bridge between the 4 'position and 2' position of the modified nucleoside" means when containing a nucleoside having a known structure of a modified bridge between the 4 'position and 2' position of the sugar, the Either one can. Further, it may further contain a base modification or another glycosyl modification.

As the bridge structure, specifically, the series is as follows. -(CR ² R ³ ) _m -O-, -(CR ² R ³ ) _m -NR ¹ -O-, -(CR ² R ³ ) _n CO-NR ¹ - or -(CR ² R ³ ) _n CO-NR ¹ -X-, here, X-based oxygen atom, sulfur atom, amine or CR ² R ³ , R ¹ -based hydrogen atom, substituted or non-substituted alkyl group, substituted or non-substituted alkenyl group, substituted Or non-substituted alkynyl, substituted or non-substituted aromatic carbocyclic, substituted or non-substituted non-aromatic carbocyclic, substituted or non-substituted aromatic heterocyclic, substituted or non-substituted non-substituted Aromatic heterocyclic group, substituted or non-substituted aromatic carbocyclic alkyl group, substituted or non-substituted non-aromatic carbocyclic alkyl group, substituted or non-substituted aromatic heterocycloalkyl group or substituted or non-substituted non-aromatic heterocyclic group; R ² are each independently a hydrogen atom based, halo, cyano, alkyl of substituted or non-substituted, the substituted or non-substituted alkenyl group or the substituted or non-substituted alkynyl group; R ³ lines Each independently is a hydrogen atom, a halogen, a cyano group, a substituted or non-substituted alkyl group, a substituted or non-substituted alkenyl group, or a substituted or non-substituted alkynyl group. train 1 to m 3, n lines 0-3.

R ¹ is preferably a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aromatic carbocyclic group, a non-aromatic carbocyclic group, an aromatic heterocyclic group, a non-aromatic heterocyclic group, or an aromatic carbon. The cycloalkyl group, the non-aromatic carbocyclic alkyl group, the aromatic heterocycloalkyl group or the non-aromatic heterocycloalkyl group may have one or more substituent groups selected from the group of α.

R ² and R ³ are preferably a hydrogen atom.

The m system is preferably 1 or 2.

The n is preferably 0 or 1.

As the bridging structure, -(CR ² R ³ ) _m -O- or -(CR ² R ³ ) _n CO-NR ¹ - is preferred; here, R1 is a hydrogen atom, a substituted or non-substituted alkane Alkenyl, substituted or non-substituted alkenyl or substituted or non-substituted alkynyl; R2 each independently a hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or non-substituted alkenyl or substituted Or a non-substituted alkynyl group; R3 each independently a hydrogen atom, a halogen, a cyano group, a substituted or non-substituted alkyl group, a substituted or non-substituted alkenyl group or a substituted or non-substituted alkynyl group; m series 1 to 3 ; n is 0 to 3.

The bridging structure is particularly preferably -CH ₂ -O- or -CO-NR ¹ - (R ¹ -based hydrogen atom or alkyl group).

"Halogen" includes a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. In particular, a fluorine atom and a chlorine atom are preferred.

The "alkyl group" contains a carbon number of 1 to 15, preferably a carbon number of 1 to 10, more preferably a carbon number of 1 to 6, more preferably a carbon number of 1 to 4, or a branched hydrocarbon group. For example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl , isohexyl, n-heptyl, isoheptyl, n-octyl, isooctyl, n-decyl, n-decyl and the like.

Preferred examples of the "alkyl group" include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, t-butyl, n-pentyl. As a more preferable aspect, a methyl group, an ethyl group, a n-propyl group, an isopropyl group, and a tributyl group are mentioned.

"Alkenyl" contains more than one double bond at any position The carbon number is from 2 to 15, preferably from 2 to 10, more preferably from 2 to 6, more preferably from 2 to 4 carbon atoms, or a branched hydrocarbon group. For example, vinyl, aromatic heterocyclic, propenyl, isopropenyl, butenyl, isobutenyl, isoprenyl, butadienyl, pentenyl, isopentenyl, pentadienyl , hexenyl, isohexenyl, hexadienyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, Pentadecyl and the like.

Preferred examples of the "alkenyl group" include a vinyl group, an aromatic heterocyclic group, a propenyl group, an isopropenyl group, and a butenyl group.

The "alkynyl group" includes a carbon number of 2 to 10, preferably 2 to 8 carbon atoms, more preferably 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms, having one or more reference bonds at any position. Straight chain or branched hydrocarbon group. For example, it includes an ethynyl group, a propynyl group, a butynyl group, a pentynyl group, a hexynyl group, a heptynyl group, an octynyl group, a decynyl group, a decynyl group, and the like. These lines can also have double bonds at any position.

Preferred examples of the "alkynyl group" include an ethynyl group, a propynyl group, a butynyl group, and a pentynyl group.

The "aromatic carbocyclic group" means a monocyclic ring or a cyclic aromatic hydrocarbon group of two or more rings. For example, a phenyl group, a naphthyl group, an anthryl group, a phenanthryl group,

Preferred examples of the "aromatic carbocyclic group" include a phenyl group.

The "non-aromatic carbocyclic group" means a monocyclic ring or a cyclic saturated hydrocarbon group of 2 or more rings or a cyclic non-aromatic unsaturated hydrocarbon group. The non-aromatic carbocyclic group having two or more rings is contained in a single ring or a non-aromatic carbocyclic group having two or more rings, and the ring of the above-mentioned "aromatic carbocyclic group" is condensed.

Further, the "non-aromatic carbocyclic group" includes a bridging group as described below or a group forming a spiro ring.

The monocyclic non-aromatic carbocyclic group is preferably a carbon number of 3 to 16, more preferably a carbon number of 3 to 12, more preferably a carbon number of 4 to 8. For example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclodecyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, ring Hexenyl, cycloheptenyl, cyclohexadienyl and the like.

Examples of the non-aromatic carbocyclic group having two or more rings include a dihydroindenyl group, a fluorenyl group, an anthranyl group, a tetrahydronaphthyl group, and an anthracenyl group.

The "aromatic heterocyclic group" means a monocyclic ring having one or more of the same or different hetero atoms selected from O, S and N in the ring or an aromatic ring group having two or more rings.

The aromatic heterocyclic group having two or more rings is also one obtained by condensing a ring having the above-mentioned "aromatic carbocyclic group" in a monocyclic ring or an aromatic heterocyclic group having two or more rings.

The monocyclic aromatic heterocyclic group is preferably 5 to 8 members, more preferably 5 members or 6 members. For example, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, anthracene Base, pyrimidinyl, pyridyl Base, triazolyl, three Base, tetrazolyl, furyl, thienyl, isoxazolyl, oxazolyl, oxadiazolyl, isothiazolyl, thiazolyl, thiadiazolyl, and the like.

Examples of the 2-ring aromatic heterocyclic group include a mercapto group, an isodecyl group, a carbazolyl group, and an anthracene group. Base, quinolyl, isoquinolyl, Olinyl group, hydrazine Base, quinazolinyl, Pyridyl, quin Polinyl, fluorenyl, acridinyl, benzimidazolyl, benzisoxazolyl, benzoxazolyl, benzoxazolyl, benzisothiazolyl, benzothiazolyl, benzothiazide Azyl, benzofuranyl, isobenzofuranyl, benzothienyl, benzotriazolyl, imidazolidinyl, thiazolopyridinyl, imidazothiazolyl, pyrazinium Base, oxazole pyridyl, oxazolidine and the like.

Examples of the aromatic heterocyclic ring system having three or more rings include, for example, a carbazolyl group and an acridinyl group. Thiophene Base Thioyl, brown Base, dibenzofuranyl and the like.

The "non-aromatic heterocyclic group" means a monocyclic ring containing one or more identical or different hetero atoms selected arbitrarily from O, S and N in the ring or a cyclic non-aromatic ring of two or more rings. Ring base.

The non-aromatic heterocyclic group having two or more rings is contained in a monocyclic ring or an aromatic heterocyclic group having two or more rings, and has the above-mentioned "aromatic carbocyclic group" and "non-aromatic carbocyclic group". And/or the condensation of each ring in the "aromatic heterocyclic group".

Further, the "non-aromatic heterocyclic group" also includes a bridging group as described below or a group forming a spiro ring.

The monocyclic non-aromatic heterocyclic group is preferably from 3 to 8 members, more preferably 5 members or 6 members. For example, dioxanthracene, thiopyranyl, oxiranyl, propylene oxide, oxathiocyclohexane, trimethyleneimine, sulfinylene, tetrahydrothiazolyl, pyrrolidinyl, Pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolyl, pyrazolinyl, piperidinyl, piperidin Base, pyridyl ring, morpholinyl, thiopyridinyl ring, thiomorpholinyl, dihydropyridyl, tetrahydropyridyl, tetrahydrofuranyl, tetrahydropyranyl, dihydrothiazolyl, tetrahydrogen Thiazolyl, tetrahydroisothiazolyl, dihydrogen Base, hexahydroindolyl, tetrahydrodiazepine, tetrahydroanthracene Base, hexahydropyrimidinyl, two Base, two Alkyl, aziridine, dioxolinyl, oxepanyl, tetrahydrothiophenyl, thiinyl, thiazide, and the like.

Examples of the non-aromatic heterocyclic ring system having two or more rings include a porphyrin group, an isoindolyl group, a benzohydropyranyl group, an isobenzohydropyranyl group, and the like.

The "alkoxy group" means a group in which the above "alkyl group" is bonded to an oxygen atom. For example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, isobutoxy, second butoxy, pentyloxy, isovaran Oxyl, hexyloxy, and the like.

Preferred examples of the "alkoxy group" include a methoxy group, an ethoxy group, a n-propoxy group, an isopropoxy group, and a third butoxy group.

In the case of "alkylamino group", it includes a monoalkylamino group and a dialkylamino group.

The "monoalkylamino group" means that the above "alkyl group" is substituted with a hydrogen atom bonded to an amine nitrogen atom. For example, a methylamino group, an ethylamino group, an isopropylamino group, etc. are mentioned. More preferably, it may be, for example, a methylamino group or an ethylamino group.

The "dialkylamino group" means a group in which the above "alkyl group" is substituted with two hydrogen atoms bonded to an amine nitrogen atom. The two alkyl groups may be the same or different. For example, dimethylamino, diethylamino, N,N-diisopropylamino, N-methyl-N-ethylamino, N-isopropyl-N-ethylamine Base. Preferred examples thereof include a dimethylamino group and a diethylamino group.

The "alkylthio group" means a group in which the above "alkyl group" is bonded to a sulfur atom.

Examples of the substituent which is "substituted or non-substituted alkyl group", "substituted or non-substituted alkenyl group", or "substituted or non-substituted alkynyl group" include the following substituents. The carbon atom at any position may be bonded to one or more kinds selected from the following substituents.

Substituents: halogen, hydroxy, carboxy, amine, imine, hydroxylamine, hydroxyimino, formamidine, methyl methoxy, amine carbhydryl, amine sulfonyl, sulfonyl, sulfin Acid group, sulfo group, thiomethyl group, thiocarboxy group, dithiocarboxy group, thiocarbamyl group, cyano group, nitro group, nitroso group, azide group, sulfhydryl group, ureido group, formazan group , mercapto, trialkylcarbenyl, alkoxy, alkenyloxy, alkynyloxy, haloalkoxy, alkylcarbonyl, olefinylcarbonyl, alkynylcarbonyl, monoalkylamino, dialkylamino, alkane Base group, alkenyl fluorenyl group, alkynyl fluorenyl group, monoalkylcarbonylamino group, dialkylcarbonylamino group, monoalkyldecylamino group, dialkyldecylamino group, alkylimino group, alkenyl group Imino, alkynylimine, alkylcarbonylimido, olefincarbonylimino, alkynylcarbonylimido, alkoxyimino, alkenyloxyimido, alkynyloxyimine, alkane Carbonyloxy, olefinoxyoxy, alkynyloxy, alkoxycarbonyl, alkoxycarbonyl, alkynyloxycarbonyl, alkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, alkylsulfinyl, Alkene Sulfosyl, alkylenesulfonyl, monoalkylamine, dialkylamine, mercapto, monoalkylamine sulfonyl, dialkylamine sulfonyl, aromatic carbocyclic, Non-aromatic carbocyclic group, aromatic heterocyclic group, non-aromatic heterocyclic group, aromatic carbocyclic group, non-aromatic carbocyclic group, aromatic heterocyclic group, non-aromatic heterocyclic ring Oxy group, aromatic carbocyclic carbonyl group, non-aromatic carbocyclic carbonyl group, aromatic heterocyclic carbonyl group, non-aromatic heterocyclic carbonyl group, aromatic carbo oxycarbonyl group, non-aromatic carbo oxycarbonyl group, aromatic heterocyclic ring Oxycarbonyl, non-aromatic heterocyclic oxycarbonyl, aromatic carboalkoxy, non-aromatic carbocycloalkoxy, aromatic heterocycloalkoxy, non-aromatic heterocycloalkoxy, aromatic carbon a cycloalkoxycarbonyl group, a non-aromatic carbocyclic alkoxycarbonyl group, an aromatic heterocycloalkoxycarbonyl group, a non-aromatic heterocycloalkoxycarbonyl group, an aromatic carbocyclic alkylamino group, a non-aromatic carbocyclic alkylamino group, Aromatic heterocyclic alkylamino group, non-aromatic heterocyclic alkylamino group, aromatic carbocyclic sulfonyl group, non-aromatic carbosulfonyl group, aromatic heterocyclic ring Acyl, sulfo acyl non-aromatic heterocyclic, non-aromatic carbocyclic sulfone group, aromatic carbocyclic sulfone group, an aromatic heterocyclic sulfone group and non-aromatic heterocyclic sulfone group.

As a "substituted or non-substituted aromatic carbocyclic group", "substituted or non-substituted non-aromatic carbocyclic group", "substituted or non-substituted aromatic heterocyclic group", "replacement or non-replacement" Non-aromatic heterocyclic group, "substituted or non-substituted aromatic carbocyclic alkyl group", "substituted or non-substituted non-aromatic carbocyclic alkyl group", "substituted or non-substituted aromatic heterocycloalkyl group" And "non-aromatic carbon rings", "non-aromatic carbon rings", "aromatic heterocyclic rings" and "non-aromatic heterocyclic rings" on the ring of "substituted or non-substituted non-aromatic heterocycloalkyl" Examples of the substituent group include the following substituents. Ring The atom at any position may be bonded to one or more groups selected from the following substituents.

Substituents: halogen, hydroxy, carboxy, amine, imine, hydroxylamine, hydroxyimino, formamidine, methyl methoxy, amine carbhydryl, amine sulfonyl, sulfonyl, sulfin Acid group, sulfo group, thiomethyl group, thiocarboxy group, dithiocarboxy group, thiocarbamyl group, cyano group, nitro group, nitroso group, azide group, sulfhydryl group, ureido group, formazan group , mercapto, trialkylcarbenyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, alkenyloxy, alkynyloxy, haloalkoxy, alkoxyalkyl, alkoxy Alkoxy, alkylcarbonyl, olefincarbonyl, alkynylcarbonyl, monoalkylamino, dialkylamino, alkyl fluorenyl, alkenyl fluorenyl, alkynyl fluorenyl, monoalkylcarbonylamino, dialkylcarbonylamine Base, monoalkyldecylamino, dialkyldecylamino, alkylimino, alkenylimine, alkynylene, alkylcarbonylimido, olefincarbonylimido, alkynylcarbonyl Imino, alkoxyimino, alkenyloxyimido, alkynyloxyimido, alkylcarbonyloxy, olefincarbonyloxy, alkynyloxy, alkoxycarbonyl, alkoxycarbonyl, alkyne Carbonyl, alkylsulfonyl, alkenyl Mercapto, alkynylsulfonyl, alkylsulfinyl, enesulfinyl, alkynesulfinyl, monoalkylamine, dialkylamine, mercapto, monoalkylamine sulfonyl , dialkylamine sulfonyl, aromatic carbocyclic, non-aromatic carbocyclic, aromatic heterocyclic, non-aromatic heterocyclic, aromatic carboepoxy, non-aromatic carbon Epoxy group, aromatic heterocyclic oxy group, non-aromatic heterocyclic oxy group, aromatic carbocyclic carbonyl group, non-aromatic carbocyclic carbonyl group, aromatic heterocyclic carbonyl group, non-aromatic heterocyclic carbonyl group, aromatic carbon epoxy a carbonyl group, a non-aromatic carbocyclic oxycarbonyl group, an aromatic heterocyclic oxycarbonyl group, a non-aromatic heterocyclic oxycarbonyl group, an aromatic carbocyclic alkyl group, a non-aromatic carbocycloalkyl group, Aromatic heterocycloalkyl group, non-aromatic heterocycloalkyl group, aromatic carbocycloalkoxy group, non-aromatic carbocycloalkoxy group, aromatic heterocycloalkoxy group, non-aromatic heterocycloalkoxy group, aromatic Group carbocyclic alkoxycarbonyl, non-aromatic carbocyclic alkoxycarbonyl, aromatic heterocycloalkoxycarbonyl, non-aromatic heterocycloalkoxycarbonyl, aromatic carboalkoxyalkyl, non-aromatic carbocycloalkoxy Alkyl group, aromatic heterocycloalkoxyalkyl group, non-aromatic heterocycloalkoxyalkyl group, aromatic carbocyclic alkylamino group, non-aromatic carbocyclic alkylamino group, aromatic heterocycloalkyl group Amine, non-aromatic heterocyclic alkylamino group, aromatic carbocyclic sulfonyl group, non-aromatic carbosulfonyl group, aromatic heterocyclic sulfonyl group, non-aromatic heterocyclic sulfonyl group, non-aromatic A carbocyclic fluorenyl group, an aromatic carbocyclic fluorenyl group, an aromatic heterocyclic fluorenyl group, and a non-aromatic heterocyclic fluorenyl group.

The alkyl group of "aromatic carbocycloalkyl", "non-aromatic carbocycloalkyl", "aromatic heterocycloalkyl", and "non-aromatic heterocycloalkyl" is also the same as the above "alkyl" .

The "aromatic carbocycloalkyl group" means an alkyl group obtained by replacing one or more of the above "aromatic carbocyclic groups". For example, benzamidine, phenethyl, phenylpropynyl, benzhydryl, trityl, naphthylmethyl, a group shown below, etc. are mentioned.

Preferred examples of the "aromatic carbocyclic alkyl group" include benzamidine, phenethyl and diphenylmethyl.

The "non-aromatic carbocyclic alkyl group" means an alkyl group obtained by replacing one or more of the above "non-aromatic carbocyclic groups". Further, the "non-aromatic carbocyclic alkyl group" also includes a "non-aromatic carbocyclic alkyl group" in which the alkyl moiety is replaced by the above "aromatic carbocyclic group". For example, a cyclopropylmethyl group, a cyclobutylmethyl group, a cyclopentylmethyl group, a cyclohexylmethyl group, a group shown below, etc. are mentioned.

The "aromatic heterocycloalkyl group" means an alkyl group substituted with one or more of the above "aromatic heterocyclic groups". Further, the "aromatic heterocycloalkyl group" also includes an "aromatic heterocycloalkyl group" in which the alkyl moiety is substituted with the above "aromatic carbocyclic group" and/or "non-aromatic carbocyclic group". For example, pyridylmethyl, furylmethyl, imidazolylmethyl, decylmethyl, benzothiophenylmethyl, oxazolylmethyl, isoisoxazolylmethyl, thiazolyl A methyl group, an isothiazolylmethyl group, a pyrazolylmethyl group, an isopyrazolylmethyl group, a pyrrolidinylmethyl group, a benzoxazolylmethyl group, a group shown below, etc.

The "non-aromatic heterocycloalkyl group" means an alkyl group substituted with one or more of the above "non-aromatic heterocyclic groups". Further, the "non-aromatic heterocycloalkyl group" also includes an alkyl moiety which is substituted by the above "aromatic carbocyclic group", "non-aromatic carbocyclic group" and/or "aromatic heterocyclic group". "Non-aromatic heterocycloalkyl". For example, tetrahydropyranylmethyl, pyridylcycloethyl, hexahydropyridylmethyl, piperidine A methyl group, a group shown below, or the like.

The anti-intentional oligonucleotide (or a modified form thereof) of the present invention can be synthesized by a usual method, and for example, a commercially available nucleic acid automatic synthesizing apparatus (for example, manufactured by Applied Biosystems, Inc., Japan) ) to easily synthesize. The synthesis method includes a solid phase synthesis method using phosphoniumamine, a solid phase synthesis method using hydrogenphosphonate, and the like. For example, it is disclosed in Tetrahedron Letters 22, 1859-1862 (1981) International Publication No. 2011/052436, and the like.

The anti-intentional oligonucleotide of the present invention comprises any biologically active metabolite or residue (directly, or indirectly) when administered to an animal including humans. Pharmaceutically acceptable salts, esters, or salts of related esters, or any other equivalent. That is, a prodrug comprising a counter-oligonucleotide of the present invention, a pharmaceutically acceptable salt, a pharmaceutically acceptable salt of the prodrug, and other biological equivalents.

"Precursor" means an inactive form or a lower activity form that utilizes the action of an intrinsic enzyme or other chemical substance and/or changes its state into its active form (ie, drug) in its body or within a cell. Precursor. The precursor drug of the anti-intentional oligonucleotide of the present invention can be in accordance with international Modulation is described in the publication of No. 93/24510, International Publication No. 94/26764.

"Pharmaceutically acceptable salt" means a physiologically and pharmaceutically acceptable salt of the anti-intentional oligonucleotide of the present invention, i.e., to maintain the desired biological activity of the anti-infectious oligonucleotide, It does not impart a salt to the effects of unwanted toxicology.

Examples of the pharmaceutically acceptable salt include alkali metals (for example, lithium, sodium, potassium, etc.), alkaline earth metals (for example, calcium, barium, etc.), magnesium, and transition metals (for example, zinc, iron, etc.). , ammonia, organic bases (eg, trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, meloamine, diethanolamine, ethylidene diamine, pyridine, methylpyridine , quinoline, etc.) and a salt of an amino acid, or with a mineral acid (for example, hydrochloric acid, sulfuric acid, nitric acid, carbonic acid, hydrogen bromide, phosphoric acid, hydrogen iodide, etc.), and an organic acid (for example, formic acid, Acetic acid, propionic acid, trifluoroacetic acid, citric acid, lactic acid, tartaric acid, oxalic acid, maleic acid, fumaric acid, phenylglycolic acid, glutaric acid, malic acid, benzoic acid, citric acid, ascorbic acid, benzenesulfonic acid, p-toluene a salt of acid, methanesulfonic acid, ethanesulfonic acid, or the like. In particular, it may, for example, be a salt with hydrochloric acid, sulfuric acid, phosphoric acid, tartaric acid or methanesulfonic acid. These salts can be formed by a conventionally carried out method.

The invention also encompasses pharmaceutical compositions comprising the anti-intelligic oligonucleotides of the invention. The administration method and preparation of the pharmaceutical composition of the present invention can be used as long as it is a preparation method and preparation known in the art. The administration method and formulation of the anti-intelligent oligonucleotide are also disclosed, for example, in the following documents.

International Publication No. 2004/016749, International Publication No. 2005/083124, International Publication No. 2007/143315, International Publication No. 2009/071680, and the like.

The pharmaceutical composition of the present invention can be administered by various methods depending on whether it is desired to be a local or systemic treatment or in the field of treatment. The administration method may be, for example, partial (eye, intravaginal, intrarectal, intranasal, transdermal), oral, or non-oral. For non-oral administration, it can be administered by intravenous injection or by drip, subcutaneous, intraperitoneal or intramuscular injection, suction or inhalation for pulmonary administration, subdural administration, intraventricular administration. Vote and so on.

When the pharmaceutical composition of the present invention is administered topically, a preparation such as a transdermal patch, an ointment, a lotion, a cream, a gel, a drop, a suppository, a spray, a liquid, a powder or the like can be used.

The composition for oral administration may, for example, be a powder, granules, water or a suspension dissolved in a non-aqueous solvent or a solution, a capsule, a powder, a tablet or the like.

Examples of the composition for parenteral administration, subdural space, or intraventricular administration include a sterile aqueous solution containing a buffer, a diluent, and other suitable additives.

The pharmaceutical composition of the present invention may be mixed with an excipient, a binder, a wetting agent, a disintegrating agent, a lubricant, a diluent, etc., which are suitable for the dosage form of the anti-intentional oligonucleotide of the present invention, as needed. Additives to get. In the case of an injection, it may be a preparation formed by sterilization treatment together with an appropriate carrier.

The excipients may, for example, be lactose, white sugar, glucose, starch, calcium carbonate or crystalline cellulose. Examples of the binder include methyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, gelatin or polyvinylpyrrolidone. Examples of the disintegrating agent include carboxymethylcellulose, sodium carboxymethylcellulose, starch, sodium alginate, acacia powder, or sodium lauryl sulfate. As the lubricant, talc, magnesium stearate or polyethylene glycol can be used. As the base of the suppository, cocoa butter, polyethylene glycol or methyl cellulose or the like can be used. Further, a dissolution aid, a suspending agent, an emulsifier, a stabilizer, a preservative, an isotonic agent which are usually used when being prepared as a liquid preparation or an emulsion or suspension injection can be suitably added. Wait. In the case of oral administration, a flavoring agent, a fragrance, or the like may be added.

The severity of the administration is related to the severity of the condition being treated, and the duration of treatment is from a few days to a few months, either until a cure is achieved, or until the condition has subsided. The optimum feeding plan can be calculated from the measurement of the accumulation of the drug in the living body. If you have expertise in the field, you can determine the most appropriate amount, the method of investment, and the frequency of repetition. The most suitable amount varies depending on the relative potency of the respective counter-oligonucleotides, but in general, it can be calculated based on the IC50 or EC50 of the animal experiments in vitro and in vivo. For example, the amount of lipid expressed in mg/kg when the molecular weight (derived from the reverse oligonucleotide sequence and chemical structure) of the antisense oligonucleotide is administered, for example, the amount of the effect of administering the IC50 (derived from the experiment) It is calculated according to the general rules.

Since the pharmaceutical composition of the present invention has ACSL1 expression inhibitory activity, it can be used for prevention or treatment of ACSL1 related diseases. way.

Examples of diseases associated with ACSL1 include obesity (including weight management for obesity), obesity-related diseases, diabetes (especially type II diabetes), X syndrome, palpitary vascular disease, or cancer (breast cancer, colon cancer, large intestine). Cancer, ovarian cancer, lung cancer, etc.).

"obesity-related diseases" refer to diseases that are caused by obesity, or caused by obesity, or as a result of obesity. Examples of obesity-related diseases include overeating, hypertension, impaired glucose tolerance, diabetes, metabolic syndrome, abnormal lipid metabolism, arteriosclerosis, hyperuricemia, gout, fatty liver, proteinuria, obesity and kidney disease. Endometrial cancer, breast cancer, prostate cancer, colon cancer, non-inflammatory osteoarthrosis, low back pain, lumbar vertebrae, obstructive sleep apnea syndrome, coronary artery disease (myocardial infarction, angina, etc.) Disease), cerebral infarction, cerebral thrombosis, transient ischemic attack, menstrual abnormalities, Puwei's syndrome, Fröhlich syndrome, Pickwickian Syndrome, etc. The pharmaceutical composition of the present invention also has a reduced risk of left ventricular hypertrophy and the like, and a reduced risk of secondary results of obesity.

The pharmaceutical composition of the present invention can be used for the prevention or treatment of, in particular, obesity or type II diabetes.

When the pharmaceutical composition of the present invention is used for the prevention or treatment of obesity, it may be combined with one or more of various known anti-obesity agents (medical compositions containing a compound having an anti-obesity effect, It can be used in combination with drugs for weight management such as obesity or obesity. Further, the administration therapy of the pharmaceutical composition of the present invention can be combined with known dietary therapies and drugs. Combination therapy, exercise, etc.

For example, the following methods are also within the scope of the invention.

The prevention or treatment of obesity or obesity-related diseases, or the method of weight management of obesity, is characterized by being used together with the pharmaceutical composition of the present invention to administer a known anti-obesity agent.

The prevention or treatment of obesity or obesity-related diseases, or the method of weight management of obesity, characterized in that a known anti-obesity is administered to a patient who is treated with the administration of the pharmaceutical composition of the present invention for prevention or treatment. medicine.

As a known anti-obesity agent, a pharmaceutical composition containing a compound having an appetite suppressing action (selective serotonin reuptake inhibitor, etc.) and a compound having a digestive absorption inhibitory action of nutrients (α-grape) may be mentioned. Glycosidase inhibitors; SGLT-2 inhibitors, etc.), compounds with fat absorption inhibition (lipase inhibitors; bile adsorption resins, etc.), 5HT transport inhibitors, NE transport inhibitors, CB-1 antagonists/ Anti-agonist, ghrelin antagonist, H3 antagonist/anti-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 agonist, leptin precursor, opioid antagonist, orexin antagonist, BRS3 agonist, CCK -A agonist, CNTF, CNTF agonist, CNTF precursor, GHS agonist, 5HT2C agonist, Mc4r agonist, monoamine reuptake inhibitor, GLP-1 agonist, UCP-1, 2 and 3 active agents, β3 agonists, thyroid hormone β agonists, PDE inhibitors, FAS inhibitors, DGAT1 inhibitor, DGAT2 inhibitor, ACC2 inhibitor, A glucocorticoid antagonist, an ethenyl-epithein, a fatty acid transporter inhibitor, a dicarboxylic acid transporter inhibitor, and the like.

When the pharmaceutical composition of the invention is used for the prevention or treatment of type II diabetes, it may be used in combination with one or more of the known therapeutic agents for type II diabetes.

As a therapeutic agent for type II diabetes, a pharmaceutical composition containing an insulin secretion-promoting drug (for example, a thiourea (SU) drug) or a fast-acting insulin secretion-promoting drug (for example, propylbenzene) may be mentioned. Amino acid precursor drug), a glucose absorption inhibitor (for example, an α-glucosidase inhibitor (αGI drug)), an insulin resistance improving drug (for example, a biguanide agent (BG drug), a tetrahydrothiazole precursor (TZD) Medicine)), insulin preparation, peptidyl dipeptidase IV (DPP-IV) inhibitor, GLP-1 receptor agonist, type 1 sodium-dependent glucose transporter (SGLT1) inhibitor, type 2 sodium-dependent glucose Transporter (SGLT2) inhibitors, etc.

When the pharmaceutical composition of the present invention is used in combination with another pharmaceutical agent, the administration period is not limited, and it can be administered simultaneously to the administration target, or it can be administered with a time difference. Further, the pharmaceutical composition of the present invention and the other pharmaceutical agent may be administered in the form of a plurality of preparations each containing the respective active ingredients, or may be administered as a single preparation containing the two active ingredients.

Detailed description of the preferred embodiment

The invention is further illustrated in the following series of examples of the invention, but the invention is not limited thereto.

Example 1: Oligonucleotide Synthesis

Oligonucleotides related to the present invention utilize Tetrahedron Letters 22, It is synthesized by the method described in 1859-1862 (1981) and International Publication No. 2011/052436.

Specifically, the oligonucleotide containing the LNA represented by the formula (a) was entrusted to Gene Design Co., Ltd. for synthesis.

(In the formula, Base is 5-methylcytosine (C), thymine (T), adenine (A) or guanine (G).)

The oligonucleotide containing the indoleamine BNA (AmNA) represented by the formula (b) was synthesized by the method described in International Publication No. 2011/052436.

(In the formula, Base is 5-methylcytosine (C), thymine (T), adenine (A) or guanine (G), and Me is methyl.)

The oligonucleotide of 10mer to 19mer containing the LNA represented by the formula (a) and the indoleamine BNA (AmNA) represented by the formula (b) is a nucleic acid automatic synthesizer (nS-8 type, (unit) large Japanese fine machine) The system was synthesized on a 0.2 μmol scale. The elongation of the chain length is carried out by standard standard operation procedures of phosphoniumamine (solid phase carrier: CPG resin; vulcanization system using DDT (3H-1, 2-Benzodithiole-3-one, 1, 1-dioxide), etc.) The oligonucleotide at the 5 ' position of the terminal is protected by the DMTr (dimethoxytrityl) group, and the 3 ' position is supported by the solid phase. Next, after removing the DMTr group by acid treatment, the target product was cleaved from the solid phase carrier by base treatment. After neutralizing with a dilute acid, the crude product obtained by distilling off the solvent was purified by colloidal filtration chromatography and reverse phase HPLC to obtain a desired product.

Further, as the Negative Control (NC) of the present invention, a sequence having a mismatch of 5 or more bases with ACSL1 was prepared and designed as shown in Table 1. In the sequence of Table 1, the upper letter indicates the LNA represented by the formula (a). The small letter indicates DNA. The internucleotide linkage oligonucleotides are all thiophosphoric acid (P=S).

Example 2: Human anti-sense oligonucleotide sequence

The anti-infectious oligonucleotide (AON) was designed to target human ACSL1 (GenBank: NM_001995, SEQ ID NO: 1). The oligonucleotide sequences are shown in Tables 2 to 6.

In the sequences of Tables 2 to 5, the upper letter indicates the LNA represented by the formula (a). The small letter indicates DNA. The internucleotide linkage oligonucleotides are all thiophosphoric acid (P=S).

In the sequence of Table 6, the upper case indicates the indoleamine BNA (AmNA) represented by the formula (b). The small letter indicates DNA. The internucleotide linkage oligonucleotides are all thiophosphoric acid (P=S).

Example 3: Mouse anti-oligonucleotide sequence

The anti-infectious oligonucleotide (AON) was designed to target mouse ACSL1 (GenBank: NM_007981, SEQ ID NO: 3). The oligonucleotide sequences are shown in Tables 7 to 12.

In the sequences of Tables 7 to 11, the uppercase letters represent the LNAs represented by the formula (a). The small letter indicates DNA. The internucleotide linkage oligonucleotides are all thiophosphoric acid (P=S).

In the sequence of Table 12, the upper case indicates the indoleamine BNA (AmNA) represented by the formula (b). The small letter indicates DNA. The internucleotide linkage oligonucleotides are all thiophosphoric acid (P=S).

Example 4: In vitro model Cell culture

The cells were cultured in an appropriate medium as described below and maintained at 37 ° C, 95 to 98% humidity and 5% CO 2 .

HepG2: Human hepatoma cell line HepG2 was cultured with DMEM High glucose (Sigma) + 10% fetal bovine serum (FBS) + Antibiotic Antimycotic Solution (10 mL/L).

HLE: Human liver cancer cell line HLE was cultured with DMEM Low Glucose (Sigma) + 10% fetal bovine serum (FBS) + Penicillin (100 units/mL) + Streptomycin (100 ug/mL).

Hepa1c1c7: Mouse hepatoma cell line Hepa1c1c7 was cultured with α-MEM (Gibco) + 10% FBS + Antibiotic Antimycotic Solution (10 mL/L).

Example 5: Evaluation of antisense oligonucleotides against ACSL1

(1) Evaluation based on changes in mRNA expression

In this example, it was confirmed that the human or mouse ACSL1 The base sequence is based on the effectiveness of the antisense oligonucleotide designed.

As described in Examples 2 and 3, antisense oligonucleotides were designed and produced, and knockdown experiments were performed on human HepG2 cells and mouse Hepa1c1c7 cells.

The knockdown experiments on human HepG2 cells and mouse Hepa1c1c7 cells were carried out using the produced reverse oligonucleotide and Negative Control (NC, SEQ ID NO: 5). In human HepG2 cells, the antisense oligonucleotide was introduced by using Lipofectamine LTX reagent (invitrogen), and was added so that the final concentration of the anti-intelligic oligonucleotide in the cell culture solution was 5 nM or 20 nM. Cellular introduction was carried out in mouse Hepa1c1c7 cells using Lipofectamine RNAiMAX reagent (invitrogen), and was added so that the final concentration of the reverse oligonucleotide in the cell culture solution was 20 nM. After 24 hours of introduction, the cells were recovered by Fastlane (QIAGEN) and quantitative PCR was performed. Use GAPDH as an intrinsic control.

In the introduction experiment using natural absorption, in the cells described in Example 4, the reverse oligonucleotide was introduced into the cells without using a reagent, so that the final concentration in the cell culture solution was 5 μM. The way to add. In human HLE cells and mouse Hepa1c1c7 cells, cells were recovered by Fastlane (QIAGEN) and quantitative PCR was performed 120 hours after introduction. Use GAPDH as an intrinsic control.

The primer sequence used to determine the amount of human ACSL1 expression was: Fw primer: GCAGCGGCATCATCAGAAAC (SEQ ID NO: 157); Rv primer: TGTCACCATCAGCCGGACTC (SEQ ID NO: 158); primer sequence used to determine the amount of human GAPDH expression: Fw primer: GCACCGTCAAGGCTGAGAAC (SEQ ID NO: 159); Rv primer: TGGTGAAGACGCCAGTGGA (SEQ ID NO: 160).

The primer sequence used to determine the expression level of mouse ACSL1 was: Fw primer: AGGTGCTTCAGCCCACCATC (SEQ ID NO: 161); Rv primer: AAAGTCCAACAGCCATCGCTTC (SEQ ID NO: 162); primer sequence used to determine the amount of expression of mouse Gapdh Use: Fw primer: TGTGTCCGTCGTGGATCTGA (SEQ ID NO: 163); Rv primer: TTGCTGTTGAAGTCGCAGGAG (SEQ ID NO: 164).

The results are shown in Tables 13, 14, 15, 16 and Fig. 1. In Table 13, regarding the reverse-inducing oligonucleotide in which the cell was introduced using the Lipofectamine RNAiMAX reagent, the ratio of the mRNA reduction of ACSL1 to the amount of untreated cells in the mouse Hepa1c1c7 cells normalized by GAPDH was expressed as knockdown. effectiveness. The NC line Negative Control in Table 13, N.D., refers to the amount of mRNA reduction of ACSL1 below the detection limit, or to increase the amount of mRNA without inhibiting ACSL1. In Table 14, regarding the reverse-inducing oligonucleotides which were subjected to cell introduction using Lipofectamine LTX reagent, the ratio of mRNA reduction of ACSL1 to the ratio of untreated cells in human HepG2 cells normalized by GAPDH was expressed as Knockdown efficiency. The NC system in Table 14 is Negative Control. In Table 15, the anti-intentional oligonucleotides in which the cells were introduced without using the reagents were expressed as knockdown in the amount of mRNA reduction of ACSL1 in the Hepa1c1c7 cells normalized by GAPDH with respect to the untreated cells. effectiveness. In Table 16, the anti-intentional oligonucleotides in which the cells were introduced without using the reagents were expressed as the knockdown efficiency of the ratio of the mRNA reduction of ACSL1 to the untreated cells in the human HLE cells normalized by GAPDH. . In Fig. 1, the knockdown efficiency of the human-induced HepG2 cells using the Lipofectamine LTX reagent for cell-introduced anti-intentional oligonucleotides (5 nM and 20 nM) is shown.

As a result, the antisense oligonucleotide of the present invention exhibits excellent knockdown activity against HepG2 cells, HLE cells, and/or Hepa1c1c7 cells as compared with other antisense oligonucleotides.

A reverse-inducing oligonucleotide for cell introduction using Lipofectamine LTX reagent or Lipofectamine RNAiMAX reagent, which exhibits a knockdown efficiency sequence of 75% or more relative to untreated cells in both HepG2 cells and Hepa1c1c7 cells. It is considered to have a sequence of heterogeneity between human and mouse, and the anti-infectious oligonucleotides (AON) number 1, 4, 5, 6, 15, 17, 18, 42, 43, 50, 51, 55 were found. , 56, 57, 58, 62, 63, 64, 65, 74, 75, 76, 77, 114, 115, 116, 117 have inter-species crossover. Furthermore, anti-initial oligonucleotides (AON) Nos. 42 and 50, 43 and 51, 55 and 62, 56 and 63, 57 and 64, 58 and 65, 74 and 114, 75 and 115, 76 and 116, 77 The sequence with the 117 line was designed to be identical to the same portion of the human and mouse ACSL1 genes, respectively. Even the same sequence in humans and mice does not necessarily show knockdown activity together, so they exhibit knockdown activity together. The above sequences are very useful in the development of new drugs.

(2) Cross-evaluation with other ACSL families

In the present example, the cross-overness of ACSL3 and ACSL5 of the ACSL family other than ACSL1 was evaluated in the knockdown effect of the anti-intelligent oligonucleotide of the present invention.

(1) A quantitative PCR was performed on the sample recovered by the method described. Use GAPDH as an intrinsic control.

The primer sequence used to determine the amount of expression of human ACSL3 was: Fw primer: ATACGGGCTCACTGAATCTGCTG (SEQ ID NO: 165); Rv primer: AGCAAACTAATGGTGCTCCCACTC (SEQ ID NO: 166); primer sequence used to determine the amount of human ACSL5 expression was used: Fw primer: GGAACTCTGAAGATCATCGACCGTA (SEQ ID NO: 167); Rv primer: CTGTGTCAGGAACCACCACTCCTA (SEQ ID NO: 168).

The primer sequence used to determine the expression level of mouse Acsl3 was: Fw primer: GCAACAACGCAGCGATTCA (SEQ ID NO: 169); Rv primer: AGCAAACTAATGGTGCTCCCACTC (SEQ ID NO: 170); primer sequence system used to determine the expression amount of mouse Acsl5 Use: Fw primer: CATTCGGCGGGACAGTTTG (SEQ ID NO: 171); Rv primer: ATCCCATTGCAGCCCTGAAG (SEQ ID NO: 172).

The results are shown in Tables 17 and 18. In Table 17, the ratio of the amount of mRNA reduction of ACSL3 or ACSL5 relative to untreated cells in mouse Hepa1c1c7 cells normalized by GAPDH was expressed as knockdown efficiency. In Table 18, it will be normalized with GAPDH. The ratio of mRNA reduction of ACSL3 or ACSL5 to untreated cells in human HepG2 cells is expressed as knockdown efficiency. Table 17 or N.D. in 18 means that the mRNA reduction of ACSL3 or ACSL5 is below the detection limit, or that the amount of mRNA is increased, but ACSL3 or ACSL5 is not inhibited.

As a result, it was confirmed that the anti-intelligence oligonucleotide of the present invention inhibits ACSL1 but does not inhibit ACSL3 and ACSL5.

(3) Evaluation based on changes in protein expression

In the present example, the knockdown activity of the antisense oligonucleotide of the present invention and the negative control (SEQ ID NO: 5) were evaluated based on the change in the expression amount of the ACSL1 protein in HepG2 cells. Further, the ACSL family specificity in HepG2 cells was evaluated for the antisense oligonucleotide of the present invention and Negative Control (SEQ ID NO: 5).

(1) The anti-infectious oligonucleotide was subjected to gene transfection in the cells by the method described, and after 48 hours, the cells were recovered by RIPA buffer (Sigma), and protein detection was carried out by Western staining. Detection of β-actin was performed as a control group.

The secondary antibody is a 1 ACSLl using rabbit anti ACSL1 antibody (Cell Signaling) , 2 secondary antibody is used ^{ECL TM Peroxidase-labeled anti-rabbit} antibody (GE).

The secondary antibody is a 1 ACSL3 using rabbit anti ACSL3 antibody (Proteintech), 2 secondary antibody is used ^{ECL TM Peroxidase-labeled anti-rabbit} antibody.

The secondary antibody is a 1 ACSL5 using rabbit anti ACSL5 antibody (Proteintech), 2 secondary antibody is used ^{ECL TM Peroxidase-labeled anti-rabbit} antibody.

1 anti-β-actin system using the mouse anti β-actin antibody (Sigma ), 2 secondary antibody is used ^{ECL TM Peroxidase-labeled anti-mouse} antibody (GE).

The results are shown in Fig. 2 and Fig. 3. In Figure 2, the knockdown effect for ACSL1 is shown. As a result, it was found that the AON numbers 1, 4, 5, 6, 43, 55, 56, and 57 were final concentrations of 5 nM, and exhibited a knockdown effect even at the protein level. In Fig. 3, the evaluation results of the intersection of ACSL3 and ACSL5 are shown. As a result, it was found that the antisense oligonucleotide of the present invention inhibits ACSL1 even at the protein level, but does not inhibit ACSL3 and ACSL5.

Example 6: Evaluation of in vivo activity in a single administration of a reverse oligonucleotide

(1) Evaluation based on changes in mRNA expression

The ANK numbers 194, 196, 197, 198, 199, 201, and 203 selected based on the evaluation of the change in the amount of mRNA expression in vitro were evaluated for the knockdown activity according to the change in the mRNA expression level of ACSL1 in the mouse liver sputum. For C57BL/6J (male 10 weeks old, Japan CLEA), will dissolve About 0.2 ml of the anti-intentional oligonucleotide solution in physiological saline (Dayusheng Food Injection, Otsuka Pharmaceutical Factory), the dosage of each mouse individual is 10 mg/kg, 20 mg/kg and 40 mg/kg, respectively. Subcutaneous administration was carried out. About 0.5 mL of whole blood and hepatic sputum tissue were taken under Semnopentyl anesthesia 3, 7 and 14 days after the administration. The RNA extract from the liver sputum was carried out using an RNeasy 96 Universal Tissue Kit (manufactured by Qiagen) in accordance with the manufacturer's recommended experimental method. 1000 ng of the obtained RNA was subjected to reverse transcription by SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (manufactured by Life Science) in accordance with a standard experimental method to obtain cDNA. Quantitative PCR was carried out using SYBR Premix Ex Taq II (manufactured by Takara Bio Inc.). Using GAPDH as an intrinsic control, the primers were used in the same way as in vitro experiments. The results are shown in Tables 19 to 21. The N/A in the table refers to the mRNA of ACSL1 not measured. The amount of mRNA of ACSL1 normalized by GAPDH indicates the ratio with respect to untreated cells.

From the results, it was confirmed that the anti-intent oligonucleotide of the present invention exhibits concentration-dependent knockdown activity even in vivo.

(2) Evaluation based on changes in protein expression

The mouse hepatic tissue to which the anti-infectious oligonucleotide (AON No. 196, 197 or 203) was administered was disrupted in 5 times the amount of RIPA Buffer (manufactured by SIGMA), and centrifuged at 20,000 g for 20 minutes to obtain The supernatant. The amount of protein contained in the supernatant was quantified using a BCA Asssay Kit (manufactured by Pierce), and 0.01 mg of protein was subjected to electrophoresis for Western Blotting. Furthermore, Western Blotting was carried out in the same manner as the in vitro experiment. The results are shown in Figures 4 and 5.

As a result, it was found that the antisense oligonucleotide of the present invention can knock down ACSL1 even at the protein level.

Example 7: Activity evaluation of in vivo (in vivo) when reverse oligonucleotides were administered repeatedly

In this example, according to the ACSL1 in the liver of the mouse The knockdown activity of the change in the amount of mRNA expression was evaluated, and the mouse line was administered with the AON No. 203 selected according to the evaluation of the change in the expression amount of mRNA in vitro. A prey-induced obese (DIO) mouse was prepared by administering C57BL/6J (male 7-week old, Japanese CLEA) for 4 weeks to a high fat fatty food (60% kcal fat: manufactured by TestDiet Co., Ltd.) for 4 weeks. The DIO mouse was dissolved in about 0.2 ml of the anti-intelligence oligonucleotide solution of AON No. 203 of physiological saline (Otsuka, Otsuka Pharmaceutical Co., Ltd.), and subcutaneous administration was performed once a week. The dosage is 5 mg/kg/week, 10 mg/kg/week, and 40 mg/kg for the first time, and 10 mg/kg/week for the first time, and the dosage is maintained for 8 weeks. Cast. Liver tissue was taken from a part of individuals on the 14th, 28th, and 56th day after the first administration, and the change in the expression of mRNA and protein was investigated in the same manner as the single administration experiment. The results regarding changes in the performance of mRNA are shown in the table. As a result, it was confirmed that the AON No. 203 can exhibit concentration-dependent knockdown activity even when it is administered in vivo (in vivo). The results of the change in the expression of the amount of protein are shown in Fig. 6.

From this result, it was confirmed that the antisense oligonucleotide of the present invention can knock down ACSL1 even at the protein level. It was found that the knockdown efficiency of mRNA and protein was approximately the same.

Example 8: Inhibition effect of weight gain when reverse oligonucleotides were administered repeatedly

The effect of increasing the body weight of the mouse when AON No. 203 was repeatedly administered was evaluated. Four weeks of high-fat fat food (60% kcal fat: manufactured by TestDiet Co., Ltd.) was administered to C57BL/6J (7-year-old male, CLEA, Japan) for 4 weeks. Prey-induced obese (DIO) mice. The average value of the body weight is divided into 7 to 8 per group, and about 0.2 ml of the anti-instantaneous oligonucleotide solution of AON No. 203 dissolved in physiological saline (大冢生食, Otsuka Pharmaceutical Factory) is performed once a week. Subcutaneous injection. The dosage is 5 mg/kg/week, 10 mg/kg/week, and 40 mg/kg for the first time, and 10 mg/kg/week for the first time, and the dosage is maintained for 8 weeks. Inject and measure the body weight and the amount of the bait. The weight change is shown in Fig. 7. Compared with the physiological saline administration group, the AON No. 203 was administered to any group, and although there was no significant change in the amount of the feeding, the weight gain suppression of any of the groups in which the AON number 203 was administered was confirm.

From the results, it was confirmed that the anti-obesity effect of ACSL1 expression in the liver sputum was suppressed by the anti-intelligence oligonucleotide of the present invention.

Example 9: Evaluation of hepatic toxicity in a single administration of anti-infected oligonucleotides

Toxicity evaluation of mouse liver sputum was performed on AON numbers 194, 198, 199, 201, and 203. Specifically, glutamate acetic acid invertase (GOT) and glutamate pyruvate converting enzyme (GPT) contained in plasma were measured as hepatotoxicity markers. For C57BL/6J (male 10 weeks old, Japan CLEA), about 0.2 ml of anti-intentional oligonucleotide solution dissolved in physiological saline (Dayusheng Food, Otsuka Pharmaceutical Factory) was administered to each mouse individual. Subcutaneous administration was carried out in such a manner that the amounts were 10 mg/kg and 20 mg/kg, respectively. After the 3, 7 and 14 days after the administration, about 0.5 mL of whole blood was taken under the anesthesia of (Somnopentyl anesthesia), and heparin was added directly to the final concentration of 0.01 U/μl (Ajinomoto Pharmaceutical Co., Ltd. ). In the plasma obtained GOT and GPT were measured by using the invertase C2 Kit Wako (manufactured by Wako Pure Chemical Industries, Ltd.) in accordance with the attached instructions. In the blood of mice administered with AON numbers 194, 198, 199, 201, and 203, no significant increase in GOT and GPT was observed compared to the physiological saline administration group.

This result confirms that the anti-intentional oligonucleotide of the present invention does not exhibit hepatotoxicity.

Industrial availability

As evident from the above examples, the anti-intelligence oligonucleotides of the present invention show that ACSL1 exhibits inhibitory activity. Therefore, the compound of the present invention is used for obesity, obesity-related diseases, diabetes (especially type II diabetes), X syndrome, palpitary vascular disorder or cancer (breast cancer, colon cancer, colon cancer, ovarian cancer, lung cancer, etc.) It is very useful in the prevention or treatment of medicine (including medicine for weight management).

<110> National University Corporation Osaka University Yanyeyi Pharmaceutical Co., Ltd.

<120> Antisense oligonucleotide against ACSL1

<130> 565625

<150> JP 2012-026989

<151> 2012-02-10

<160> 172

<170> PatentIn version 3.5

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<223> Anti-intelligent oligonucleotide

<400> 79

<210> 80

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 80

<210> 81

<211> 13

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 81

<210> 82

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 82

<210> 83

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 83

<210> 84

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 84

<210> 85

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 85

<210> 86

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 86

<210> 87

<211> 14

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 87

<210> 88

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 88

<210> 89

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 89

<210> 90

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 90

<210> 91

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 91

<210> 92

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 92

<210> 93

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 93

<210> 94

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 94

<210> 95

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 95

<210> 96

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 96

<210> 97

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 97

<210> 98

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 98

<210> 99

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 99

<210> 100

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 100

<210> 101

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 101

<210> 102

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 102

<210> 103

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 103

<210> 104

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 104

<210> 105

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 105

<210> 106

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 106

<210> 107

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 107

<210> 108

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 108

<210> 109

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 109

<210> 110

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 110

<210> 111

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 111

<210> 112

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 112

<210> 113

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 113

<210> 114

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 114

<210> 115

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 115

<210> 116

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 116

<210> 117

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 117

<210> 118

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 118

<210> 119

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 119

<210> 120

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 120

<210> 121

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 121

<210> 122

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 122

<210> 123

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 123

<210> 124

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 124

<210> 125

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 125

<210> 126

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 126

<210> 127

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 127

<210> 128

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 128

<210> 129

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 129

<210> 130

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 130

<210> 131

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 131

<210> 132

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 132

<210> 133

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 133

<210> 134

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 134

<210> 135

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 135

<210> 136

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 136

<210> 137

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 137

<210> 138

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 138

<210> 139

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 139

<210> 140

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 140

<210> 141

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 141

<210> 142

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 142

<210> 143

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 143

<210> 144

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 144

<210> 145

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 145

<210> 146

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 146

<210> 147

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 147

<210> 148

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 148

<210> 149

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 149

<210> 150

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 150

<210> 151

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 151

<210> 152

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 152

<210> 153

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 153

<210> 154

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 154

<210> 155

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 155

<210> 156

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-intelligent oligonucleotide

<400> 156

<210> 157

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Introduction

<400> 157

<210> 158

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Introduction

<400> 158

<210> 159

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Introduction

<400> 159

<210> 160

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Introduction

<400> 160

<210> 161

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Introduction

<400> 161

<210> 162

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Introduction

<400> 162

<210> 163

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Introduction

<400> 163

<210> 164

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Introduction

<400> 164

<210> 165

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Introduction

<400> 165

<210> 166

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Introduction

<400> 166

<210> 167

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 167

<210> 168

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Introduction

<400> 168

<210> 169

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Introduction

<400> 169

<210> 170

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Introduction

<400> 170

<210> 171

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Introduction

<400> 171

<210> 172

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Introduction

<400> 172

Claims (11)

A reverse oligonucleotide comprising a sequence which can be heterozygous under stringent conditions to a sequence consisting of: 95 to 109, 176 to 192, 467 of SEQ ID NO: 1. To 484, 940 to 954, 1017 to 1032, 1102 to 1116, 1176 to 1197, 1222 to 1236, 1727 to 1743, 1858 to 1873, 1946 to 1960 Bit, 2294 to 2308, 2360 to 2377, 2449 to 2469, 2605 to 2624, 2689 to 2703, 2950 to 2964, 3424 to 3438 or 3591 to 3605 . As the anti-intentional oligonucleotide of claim 1 of the patent application, it inhibits the expression of ACSL1. The anti-intentional oligonucleotide of claim 1 or 2, which is 13 to 19 bases in length. The anti-intentional oligonucleotide according to any one of claims 1 to 3, which contains one or more glycosyl-modified nucleosides having a bridging structure at the 4'-position and the 2'-position. As the anti-intentional oligonucleotide of claim 4, the bridging structure is -CH ₂ -O- or -CO-NR ¹ - (R ¹ is a hydrogen atom or an alkyl group). The anti-intentional oligonucleotide of any one of claims 1 to 5, wherein one or more of the linkages between the nucleosides is a phosphorothioate linkage. The anti-intentional oligonucleotide according to any one of claims 1 to 6, which comprises SEQ ID NO: 6 to 11, 13 to 18, 20 to 22, 24 to 30, 32, a sequence of 35 to 37, 46, 48, 49, 52 to 54, 57 to 59, 64 to 68 or 70; or contained in SEQ ID NO: 6 to 11, 13 to 18, 20 to 22, 24 to 30, 32, 35 A sequence in which one or several bases are deleted, substituted or inserted in the sequence of 37, 46, 48, 49, 52 to 54, 57 to 59, 64 to 68 or 70. The anti-intentional oligonucleotide of claim 7 is composed of the following sequences: Nos. 6 to 11, 13 to 18, 20 to 22, 24 to 30, 32, 35 to 37, 46, 48, a sequence of 49, 52 to 54, 57 to 59, 64 to 68 or 70; or in sequence numbers 6 to 11, 13 to 18, 20 to 22, 24 to 30, 32, 35 to 37, 46, 48, 49, A sequence in which a sequence of 52 to 54, 57 to 59, 64 to 68 or 70 is deleted, substituted, inserted or affixed to one or several bases. A pharmaceutical composition comprising the anti-intelligence oligonucleotide of any one of claims 1 to 8. For example, the pharmaceutical composition of claim 9 is for the prevention or treatment of an ACSL1-related disease. The pharmaceutical composition of claim 10, wherein the disease is obesity or type II diabetes.