JP7549864B2 - Fat differentiation inhibitor - Google Patents

Description

This disclosure relates to oligonucleotides that have the function of suppressing fat differentiation.

Obesity is a risk factor for diabetes, high blood pressure, arteriosclerosis, heart disease, and other conditions, and is a factor that inhibits healthy lifespan. Obesity occurs when adipocytes differentiated from adipocyte precursor cells store excess lipids and become enlarged. Adipokines secreted by adipocytes affect the risk of developing lifestyle-related diseases. Therefore, controlling adipocyte differentiation is important for suppressing obesity and preventing the progression to metabolic syndrome.

For example, Patent Document 1 discloses an obesity suppressant that contains, as an active ingredient, an antisense oligonucleotide against the MXD3 gene (a nucleic acid molecule having a sequence complementary to the mRNA encoding MXD3). Patent Document 1 also discloses 5'GATGTTGCACGTATTTACCTCCATT3' (SEQ ID NO: 12) as a specific oligonucleotide.

However, since obesity is a risk factor for the various diseases mentioned above, there is a strong demand for the development of new methods to inhibit fat differentiation.

Patent Publication 2011-55755 WO2018/151225

Kazunori Sunadome, et al., “Antagonism between the Master Regulators Of Differentiation Ensures the Discreteness and Robustness of Cell Fates”, Molecular Cell 54, 526-535, May8, 2014 Simona Iezzi et al, “Deacetylase Inhibitors Increase Muscle Cell Size by Promoting Myoblast Recruitment and Fusion through Induction of Follistatin” Developmental Cell, Vol. 6, 673-684, May, 2004 Xiao Wang et al. “Trichostatin A and vorinostat promote adipogenic differentiation through H3K9 acetylation and dimethylation” Research in Veterinary Science 126 (2019) 207-212

The problem to be solved by this invention is to provide a new agent for inhibiting fat differentiation.

Under such circumstances, the present inventors conducted extensive research and found that an oligonucleotide of a certain length having a core base sequence TXGGGTG has the ability to inhibit fat differentiation. The present invention is based on this new finding. Therefore, the present invention provides the following:
Item 1. An adipogenesis inhibitor comprising an oligonucleotide having activity of inhibiting adipogenesis, the oligonucleotide comprising a core base sequence TXGGGTG (wherein X is absent or represents A) and having a length of 7 to 18 bases.
Item 2. The adipogenesis inhibitor according to Item 1, wherein the oligonucleotide comprises a core base sequence TGGGTG.
Item 3. The adipogenesis inhibitor according to Item 1 or 2, wherein the oligonucleotide comprises a core base sequence TGGGTGGGG.
Item 4. The oligonucleotide (i-1) has a base sequence represented by SEQ ID NO: 1:
SEQ ID NO: 1: AGATTAGGGTGAGGGTGA
or (i-2) a base sequence in which one or several bases have been substituted, added or deleted in the base sequence represented by SEQ ID NO: 1.
Item 4. The fat differentiation inhibitor according to any one of items 1 to 3.
Item 5. The oligonucleotide (i-1) has a base sequence represented by SEQ ID NO: 2:
SEQ ID NO: 2: TTGGGTGGGGAA
or (i-2) a base sequence in which one or several bases have been substituted, added or deleted in the base sequence represented by SEQ ID NO: 2.
Item 5. The fat differentiation inhibitor according to any one of items 1 to 4.
Item 6. The fat differentiation inhibitor according to any one of Items 1 to 5, for application to a mammalian or avian cell or individual.

According to the present invention, a new fat differentiation inhibitor can be provided. The present inventors have found that iSN04, an oligo DNA (ODN) consisting of the base sequence 5'AGATTAGGGTGAGGGTGA3' (SEQ ID NO: 1), has a muscle differentiation promoting effect (Patent Document 2). In addition, some of the fat differentiation control factors and muscle differentiation control factors are thought to act antagonistically. Specifically, it has been reported that muscle differentiation and fat differentiation proceed mutually exclusively due to the antagonistic action of MyoD, the master control factor of muscle differentiation, and PPARγ, the master control factor of fat differentiation (Non-Patent Document 1). However, the mechanisms of muscle and fat differentiation of cells involve a wide variety of factors, and many of these mechanisms have yet to be elucidated. Furthermore, it is known that there are factors that promote both muscle and fat differentiation. Specifically, it is known that trichostatin A (TSA), a histone deacetylase inhibitor, promotes muscle differentiation (Non-Patent Document 2). On the other hand, it is also known that histone acetylation contributes to fat differentiation, and it has been reported that TSA also promotes fat differentiation (Non-Patent Document 3). As described above, it is known that histone acetylation promotes both muscle differentiation and fat differentiation. Therefore, the effect of inhibiting fat differentiation by using the oligonucleotide, which is the active ingredient of the present invention, is an effect that could not be predicted from conventional technology.

1 shows an Oil Red O staining test (optical microscope photograph) in Example 1. 1 shows the results of measuring the inhibition of fat differentiation when various oligo-DNAs were administered at various times in Example 2. 1 shows the results of measuring the expression level of PPARγ in Example 3. 1 shows the results of measuring the expression level of CEBPα in Example 3. 1 shows the results of measuring the expression level of FABP4 in Example 3. 1 shows the results of measuring the expression level of Perilipin in Example 3.

In this specification, "oligonucleotide" may be either DNA, RNA, or a DNA-RNA hybrid. Furthermore, these may be double-stranded or single-stranded, and when referring to an oligonucleotide having a certain sequence, unless otherwise specified, this is taken to comprehensively mean an oligonucleotide having a complementary sequence to that. Furthermore, these nucleic acid molecules may be circular or linear, and may be either synthetic or of biological origin.

The fat differentiation inhibitor according to one embodiment of the present invention comprises an oligonucleotide having activity of suppressing fat differentiation when applied to a cell or an individual. In the present invention, "having activity of suppressing fat differentiation" may be interpreted appropriately taking into consideration the common technical knowledge in the field to which the present invention belongs. Whether or not the agent has the ability to suppress fat differentiation can be measured and evaluated, for example, in the test shown in Example 2 (Figure 2) described below, by adding the oligonucleotide to 3T3-L1 cells from day 0 to day 10 of differentiation induction, measuring the concentration of Oil Red O, and determining whether or not the agent significantly suppresses intracellular lipid accumulation compared to the control.

In one embodiment of the present invention, the oligonucleotide having activity of suppressing fat differentiation is characterized by having a core base sequence TXGGGTG (wherein X is absent or represents A). The core base sequence is preferably TGGGTG. In a preferred embodiment of the present invention, the oligonucleotide having activity of suppressing fat differentiation includes one containing the core base sequence TGGGTGGGG.

In addition, in a typical embodiment of the present invention, the length of the oligonucleotide that is the active ingredient of the fat differentiation inhibitor is 7 to 18 bases long, and preferably 12 to 18 bases long. It is useful to use oligonucleotides with base lengths in the above range from the viewpoints of the structural strength and stability of the oligonucleotide, the efficiency of absorption into organs and cells, and the ability to reduce costs when chemically synthesizing the oligonucleotide.

In a preferred embodiment of the present invention, the oligonucleotide that is the active ingredient of the adipogenesis inhibitor is, but is not limited to, for example,
(i-1) A base sequence represented by SEQ ID NO: 1:
SEQ ID NO: 1: AGATTAGGGTGAGGGTGA
or (i-2) an oligonucleotide having a base sequence in which one or several (e.g., 1 to 3, 1 to 2, or 1) bases have been substituted, added, and/or deleted in the base sequence represented by SEQ ID NO:1.
More specifically, for example,
AGATTAGGGTGAGGGTGA SEQ ID NO: 1
AGATTAGGGTGAGGGTGT SEQ ID NO: 3
AGATAGGGTGAGGGGTGA SEQ ID NO: 4
AGGATTAGGGTGAGGGTG SEQ ID NO: 5
AGATAGGGTGAGGGTG SEQ ID NO: 6
ATTAGGGTGAGGGGTGT SEQ ID NO: 7
etc.

In another more preferred embodiment of the present invention, the oligonucleotide that is the active ingredient of the adipogenesis inhibitor is, for example,
(i-1) A base sequence represented by SEQ ID NO: 2:
SEQ ID NO: 2: TTGGGTGGGGAA
or (i-2) an oligonucleotide having a base sequence in which one or several bases have been substituted, added and/or deleted in the base sequence represented by SEQ ID NO:2.
More specifically, for example,
TTGGGTGGGGAA SEQ ID NO: 2
AAGGGTGGGGTT SEQ ID NO:8
TGGGTGGGGAAA SEQ ID NO: 9
TTGGGTGGGGAAA SEQ ID NO: 10
etc.

In an embodiment of the present invention, the above oligonucleotide, which is the active ingredient of the fat differentiation inhibitor, not only inhibits fat differentiation but also promotes muscle differentiation. Therefore, in a preferred embodiment of the present invention, the above oligonucleotide is useful because it can be used to simultaneously inhibit fat differentiation and promote muscle differentiation.

The above-mentioned oligonucleotide, which is the active ingredient of the fat differentiation inhibitor of the present invention, may be, but is not limited to, an oligonucleotide having a phosphodiester bond in which the oxygen atom of the phosphate group has been replaced with a sulfur atom (e.g., phosphorothioate bond) in order to increase resistance to nucleases. In addition, the oligonucleotide may be, but is not limited to, one extracted from an organism such as lactic acid bacteria or Escherichia coli and fragmented, or one created by chemical synthesis or gene recombination technology.

In the present invention, the above-mentioned oligonucleotide, which is the active ingredient of the present invention, may be used itself as an adipogenesis inhibitor, or may be used as a pharmaceutical composition in combination with various pharma- ceutical acceptable carriers (e.g., isotonicity agents, stabilizers, pH regulators, antioxidants, solubilizers, thickeners, preservatives, etc.).
Examples of isotonicity agents include sugars such as glucose, trehalose, lactose, fructose, mannitol, xylitol, and sorbitol; polyhydric alcohols such as glycerin, polyethylene glycol, and propylene glycol; and inorganic salts such as sodium chloride, potassium chloride, and calcium chloride.
Examples of pH adjusters include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, as well as alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates or hydrogen carbonates such as sodium carbonate, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, and bases such as trometamol.
Examples of antioxidants include sodium hydrogen sulfite, dry sodium sulfite, and sodium pyrosulfite.
Examples of the solubilizing agent include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, D-mannitol, and the like.
Examples of thickening agents include polyethylene glycol, methyl cellulose, ethyl cellulose, carmellose sodium, xanthan gum, sodium chondroitin sulfate, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, and polyvinyl alcohol.
Examples of preservatives include sorbic acid, potassium sorbate, paraoxybenzoic acid esters such as methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and butyl paraoxybenzoate, chlorhexidine gluconate, quaternary ammonium salts such as benzalkonium chloride, benzethonium chloride, and cetylpyridinium chloride, alkylpolyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, and chlorhexidine.
In addition, the pharmaceutical composition may further contain a compound known to have an adipogenesis inhibitory effect in addition to the oligonucleotide. Examples of compounds known to have an adipogenesis inhibitory effect include ursolic acid, curcumin, berberine, etc. When using a compound known to have an adipogenesis inhibitory effect, the ratio of the oligonucleotide to the compound known to have an adipogenesis inhibitory effect is not particularly limited, but for example, 0.01 to 100 moles per mole of the former is preferable, and 0.1 to 10 moles is more preferable.
In an embodiment of the pharmaceutical composition, the content of the oligonucleotide in the composition is not particularly limited and can be appropriately set from conditions such as 90% by mass or more, 70% by mass or more, 50% by mass or more, 30% by mass or more, 10% by mass or more, 5% by mass or more, and 1% by mass or more.
The content of the oligonucleotide of the present invention in the formulation varies depending on the route of administration, the age, weight, symptoms, etc. of the patient and cannot be generally defined, but it is sufficient that the daily dose of the oligonucleotide is usually about 10 to 5000 mg, more preferably about 100 to 1000 mg. When administered once a day, this amount needs to be contained in one formulation, and when administered three times a day, one third of this amount needs to be contained in one formulation.
The fat differentiation inhibitor of the present invention can be formulated into injections, liniments, tablets, capsules, syrups, suppositories, etc., by mixing with various commonly used carriers and excipients, and according to known methods, but is not limited to these. The fat differentiation inhibitor can be administered orally, intravenously, intramuscularly, intraarterially, intramedullary, intrathecally, intraventricularly, percutaneously, subcutaneously, intraperitoneally, enterally, topically, sublingually, or rectally, but is not limited to these.

The fat differentiation inhibitor of the present invention can be applied to cells constituting an individual as well as to cells cultured on a medium. In addition, the fat differentiation inhibitor can be targeted to mammals, including mice and humans, or birds, including chickens, but is not limited thereto.

Specific embodiments of the present invention will be described below using examples, comparative examples, and reference examples, but the present invention is not limited to these specific embodiments.

The oligo DNA samples used in the following experiments were synthesized by replacing the phosphodiester bonds between nucleotides with phosphorothioate bonds to increase resistance to nucleases, and then purified by HPLC.

The base sequence of the oligo DNA used in the following examples is as follows:

Example 1
We used mouse embryonic fibroblast cell line 3T3-L1 (JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition, No. JCRB9014), which is a model of adipose precursor cells. First, 3T3-L1 cells were seeded on a 6-well plate, and 48 hours after reaching confluence, iSN04 was added at a final concentration of 30 μM to induce adipogenesis. During the differentiation induction period, the culture medium was replaced every two days, and the same concentration of iSN04 was added each time. On the 10th day after differentiation induction, lipid droplets in the cells were stained with Oil Red O to evaluate adipogenesis. The results (optical microscope photographs) are shown in Figure 1. Compared to the control (a sample to which an equal amount of pure water was added as iSN04), it was confirmed that adipogenesis was clearly suppressed in the sample to which iSN04 was added.

Example 2
(ODN administration period and timing)
3T3-L1 cells were seeded in a 24-well plate, and 48 hours after reaching confluence, three types of ODN, iSN04, iMyo01, or iMyo03, were added at a final concentration of 30 μM to induce adipogenesis. During the differentiation induction period, the culture medium was replaced every two days, and the same concentration of ODN was added each time. On the 10th day after differentiation induction, intracellular lipid droplets were stained with Oil Red O, and then the intracellular Oil Red O was extracted with 300 μL of isopropanol. The concentration of Oil Red O in the extract was measured using a spectrophotometer, and the amount of lipid accumulated in the cells was quantified. The results are shown in Figure 2.

In Figure 2, the label dm-n on the right side indicates the start date of ODN addition (differentiation induction start date is d0) and the end date of addition. For example, d0-4 indicates a sample in which differentiation was induced under conditions in which ODN was added from day 0 to day 4 of differentiation induction, and Oil Red O was measured on day 10.
In the groups administered iMyo01, iMyo03, or iSN04 for 10 days (d0-10), it was confirmed that the amount of lipids accumulated in 3T3-L1 cells was suppressed to 40% or less compared to the control group (group administered pure water for 10 days). These ODNs contain the base sequence TxGGGTG (x=A or -).
It was confirmed that lipid accumulation was suppressed to about 10% in iMyo01 and iMyo03 compared to the control. These ODNs contain the base sequence TGGGTGGGG.
Furthermore, we performed an experiment to confirm the timing and number of days of iSN04 administration. In the groups in which iSN04 was administered for only 4, 6, or 8 days from the first day of differentiation induction (d0-4, d0-6, d0-8), lipid accumulation was suppressed to the same extent as in the group in which iSN04 was administered for 10 days (d0-10). However, the inhibitory effect in the d0-2 group was lower than that in the d0-4, d0-6, d0-8, and d-10 groups. Furthermore, no inhibitory effect on lipid accumulation was observed in the groups in which iSN04 was administered during other periods.
These results indicate that administration of iSN04 for 4 days (d0-4) during the early stages of differentiation is sufficient to suppress lipid accumulation. In this differentiation induction system, 3T3-L1 cells first differentiate into adipocytes, and the differentiated adipocytes accumulate lipids and form lipid droplets. Lipid droplets are observed from about the 6th day of differentiation induction, but administration of iSN04 after this time does not suppress lipid accumulation. From the above, it is considered that iSN04 does not directly inhibit lipid accumulation, but suppresses the differentiation of 3T3-L1 cells into adipocytes. In the groups administered iSN04 for 4 days during the early stages of differentiation (d0-4, d0-6, d0-8, d0-10), differentiation into adipocytes capable of storing lipids was suppressed (the number of adipocytes themselves decreased), and this is thought to be reflected in the value of Oil Red O.

Example 3
(Suppression of specific gene expression)
We investigated the expression changes of adipogenic marker genes in 3T3-L1 cells induced to differentiate into adipogenic cells by administration of iSN04. 3T3-L1 cells were seeded on a 3 cm dish, and 48 hours after reaching confluence, iSN04 was administered at a final concentration of 30 μM to induce adipogenic differentiation. During the differentiation induction period, the culture medium was replaced every two days, and the same concentration of iSN04 was added each time. Total RNA of 3T3-L1 cells was collected 0, 2, 4, 6, and 10 days after differentiation induction and subjected to quantitative PCR. The expression levels of PPARγ (gene name Pparg), a master transcription factor of adipogenic differentiation, CEBPα (gene name Cebpa), a transcription factor that interacts with PPARγ, fatty acid binding protein FABP4 (gene name Fabp4), and lipid droplet surface protein Perilipin (gene name Plin1), were corrected by dividing them by the expression level of the internal standard Ywhaz gene, and the results are shown in Figures 3 to 6. The experiment was performed three times, and significant differences (*p < 0.05, **p < 0.01) were observed.
Compared to the control (a sample administered with an equal amount of pure water as iSN04), the gene expression of PPARγ, CEBPα, FABP4, and Perilipin was confirmed to be significantly decreased in the iSN04-administered group at the time points marked with *.
These results suggest that iSN04 suppresses adipogenesis through the downregulation of adipose-specific genes, ultimately inhibiting maturation of adipocytes with lipid droplets.

The oligonucleotide, which is the active ingredient of the present invention, suppressed the adipogenesis of 3T3-L1 cells. Therefore, the oligonucleotide, which is the active ingredient of the present invention, is promising as a nucleic acid material that exhibits anti-obesity effects, and is expected to be applied to supplements, etc.

Claims (2)

An adipogenesis inhibitor comprising an oligonucleotide having an activity of inhibiting adipogenesis,
(i) a base sequence represented by SEQ ID NO: 1:
SEQ ID NO: 1: AGATTAGGGTGAGGGTGA
Consists of
(ii) a base sequence represented by SEQ ID NO: 2:
SEQ ID NO: 2: TTGGGTGGGGAA
or
(iii) a base sequence represented by SEQ ID NO: 11:
SEQ ID NO: 11: TTGGGTGAGGGGAA
An adipogenesis inhibitor consisting of : The fat differentiation inhibitor according to claim 1 , which is to be applied to mammalian or avian cells or individuals.