WO2012002328A1 - Blood hdl level increasing agent or anti-arteriosclerotic agent - Google Patents

Abstract

Disclosed are: a novel PI polyamide which is useful for increasing a blood HDL level; and an anti-arteriosclerotic agent comprising the PI polyamide as an active ingredient. PI polyamide compounds (three types) which can control the expression of human ABCA1 gene are synthesized, and an anti-arteriosclerotic agent comprising any one of the PI polyamide conmpounds as an active ingredient can be produced.

Description

Blood HDL increasing agent or anti-arteriosclerosis agent

The present invention relates to a novel pyrrole imidazole polyamide (hereinafter referred to as PI polyamide), a blood high-density lipoprotein increasing agent (hereinafter referred to as blood HDL increasing agent) or an anti-arteriosclerotic agent containing the PI polyamide as an active ingredient. .

HDL, which is one of lipoproteins, is known to exhibit an anti-arteriosclerotic action, with its blood level inversely correlated with the frequency of arteriosclerotic diseases. Therefore, various substances that raise blood levels of HDL have been developed for the purpose of treating or preventing arteriosclerotic diseases such as atherosclerosis (see, for example, Patent Document 1).

ABCA1 (ATP binding cassette A1) is a protein that plays a major role when HDL extracts cholesterol from cells in peripheral tissues, and the expression level is correlated with the blood level of HDL. In view of this, a hypoHDLemia ameliorating agent and an arteriosclerosis prophylactic / therapeutic agent having a cysteine protease inhibitor that significantly and continuously increases the expression level of ABCA1 as an active ingredient have been developed (for example, see Patent Document 2). Moreover, the ABCA1 stabilizer which used the bisphenol type compound as an active ingredient is developed (for example, patent document 3). Although these are useful for raising the blood level of HDL, it has been desired to provide a blood HDL increasing agent that is more stable and effective.

Recently, in the gene expression control technology, PI polyamides that are excellent in in vivo stability and transferability to tissues and cells have attracted attention. The inventors have focused on this substance and are working on the development of a new PI polyamide that is effective for raising the blood level of HDL by an original method. As a result, the inventors of the present invention have provided a blood HDL increasing agent and the like that are more stable and effective than the conventional hypoHDL ameliorating agent and arteriosclerosis preventing / treating agent.

Special table 2009-522293 International Publication No. 2003/033023 Pamphlet International Publication No. 2005/0697904 Pamphlet

An object of the present invention is to provide a novel PI polyamide useful for increasing the blood level of HDL. Furthermore, this invention makes it a subject to provide the anti-arteriosclerosis agent which uses this PI polyamide as an active ingredient.

As a result of diligent research to solve the above problems, the present inventors have increased the expression level of ABCA1 that correlates with the blood level of HDL. Seed) was synthesized to complete the present invention.
These PI polyamides synthesized by the present inventors bind to the AP2 binding site, which is a negative transcription control region present in the human ABCA1 gene promoter region. These PI polyamides can increase the expression of the human ABCA1 gene by inhibiting the binding of AP2 to the AP2 binding site. The present inventors have confirmed that these PI polyamides increase the expression of the ABCA1 gene in in vitro and in vivo tests. It has also been confirmed that these PI polyamides increase blood HDL in in vivo studies.
Therefore, blood HDL can be increased by administering a blood HDL increasing agent containing these PI polyamides as an active ingredient to animals such as mice and humans. It is also possible to treat or prevent arteriosclerotic diseases by administering anti-atherosclerotic agents containing these PI polyamides as active ingredients to animals such as mice and humans.

That is, the present invention relates to the following (1) to (5) PI polyamide, an agent for increasing blood HDL containing the PI polyamide as an active ingredient, an anti-arteriosclerosis agent, and the like.
(1) A blood HDL increasing agent comprising one or more PI polyamides of chemical formulas 1 to 3 as an active ingredient.

(2) An anti-arteriosclerotic agent containing one or more PI polyamides of chemical formulas 1 to 3 as an active ingredient.
(3) The PI polyamide described in Chemical Formula 1 or 2.
(4) A method for increasing blood HDL, comprising a step of administering the blood HDL increasing agent according to (1) to an animal.
(5) A method for treating or preventing an arteriosclerotic disease comprising the step of administering the anti-arteriosclerotic agent of (1) above to an animal.

The anti-atherosclerotic agent comprising PI polyamide provided by the present invention as an active ingredient is widely used as a drug having a new action that increases the expression of ABCA1 gene and increases HDL in the prevention and treatment of arteriosclerotic diseases. Available.

It is the figure which showed the base sequence of the human ABCA1 gene promoter region (Example 1). It is the figure which showed the base sequence of the site | part centering on the AP2 binding site which PI polyamide recognizes (Example 1). It is the figure which showed the refinement | purification result of PI polyamide (Example 1). It is the figure which confirmed the arrangement | sequence specificity of PI polyamide (Test Example 1). It is the figure which showed the effect of PI polyamide with respect to the expression of ABCA1 gene (Test Example 2). It is the figure which showed the effect of PI polyamide with respect to the expression of ABCA1 gene (Test Example 2). It is the figure which showed the effect of PI polyamide with respect to drawing | extracting out of a lipid to a cell (Test Example 3). It is the figure which showed the effect of PI polyamide with respect to drawing | extracting out of a lipid to a cell (Test Example 3). It is the figure which showed the effect of PI polyamide with respect to the expression of ABCA1 protein (Test Example 4). It is the figure which confirmed the arrangement | sequence specificity of PI polyamide (Test Example 5). It is the figure which showed the effect of PI polyamide with respect to the expression of ABCA1 gene (Test Example 5). It is the figure which showed the effect of PI polyamide with respect to drawing | extracting out lipid (free cholesterol) outside a cell (Test Example 5). It is the figure which showed the effect of PI polyamide with respect to extraction of the lipid (phospholipid) out of the cell (Test Example 5). It is the figure which showed the effect of PI polyamide in the blood HDL increase action in vivo (Test Example 5).

The “blood HDL increasing agent” of the present invention refers to an agent that can increase blood HDL by administration to mammals such as humans by injection or the like.
The “blood HDL increasing agent” of the present invention may be any agent that contains one or a combination of PI polyamides of Chemical Formulas 1 to 3 as an active ingredient, and includes a pharmaceutically acceptable carrier in addition to the active ingredient. May be.
The PI polyamides of the chemical formulas 1 to 3 can be synthesized by any conventionally known method for synthesizing PI polyamides, but it is particularly preferred to synthesize them by the PSSM-8 synthesis program.

These PI polyamides can be used as active ingredients of anti-arteriosclerotic agents. The “anti-arteriosclerotic agent” refers to an agent for the purpose of preventing or treating arteriosclerotic diseases, and any agent may be used as long as the active ingredient is one or a combination of PI polyamides of the chemical formulas 1 to 3, In addition to the active ingredient, it may contain a pharmaceutically acceptable carrier.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

<Preparation of PI polyamide>
1. Design of PI polyamide The following 1 to 3 PI polyamides (3 types) were designed to bind to a site centering on the AP2 binding site of the human ABCA1 gene promoter region. FIG. 1 shows the base sequence of the human ABCA1 gene promoter region. In FIG. 1, the portion surrounded by a blue frame and described as the AP2 binding site at the top is the base sequence of the AP2 binding site. Hereinafter, each symbol in the chemical formula has the following meaning (Ac: acetyl, Py: pyrrole, Im: imidazole, β: β-alanine, γ: γ-butyric acid, Dp: N, N-dimethyl-1,3-propane Diamine).
PI polyamide enables recognition of a DNA double helix structure by combining two pairs, such as a combination of Py / Py and Py / Im. However, a PI polyamide composed of a combination of Py / Py and Py / Im is structurally very rigid, and when five or more are connected, they are disengaged from the minor groove of the DNA double helix structure, and the binding force decreases. Therefore, unless a flexible β-alanine pair (a combination of β / β) that recognizes W (A or T) is introduced every third, there is a major limitation that it cannot bind to the minor groove of the DNA double helix structure. is there. For this reason, it has been difficult to construct PI polyamides that can recognize base sequences rich in GC sequences. Although a PI polyamide that recognizes a 4-8 base sequence has been obtained, it requires at least 12 bases to recognize a unique gene on the genome. There was a need to provide a recognizable PI polyamide.
In the present invention, the automatic synthesis of PS polyamide that recognizes 12 bases was succeeded for the first time by using the automatic synthesizer PSSM-8 and optimizing it. In the present invention, in the design of PI polyamide, a hairpin structure is incorporated into the polyamide so that the two pairs of polyamides are not displaced. In addition to this, in addition to the conventional Py / Py and Py / Im combinations, β-alanine that keeps a high degree of freedom in combination with Py or Im and flexibly responds to the bending of the spiral structure of the DNA double helix. Was adopted. That is, in the present invention, a structure in which β-alanine crosses by adding a combination of Py / β and Im / β is added. This eliminates the need for W (A or T) sequence recognition rules based on β / β β-alanine combinations for every 3 bases, and provides GC, CG, etc. It was possible to construct a PI polyamide capable of recognizing the base sequence. That is, the present invention is the first to obtain a hairpin type PI polyamide that can enhance the selectivity of recognition of a base sequence rich in GC sequences and can be bonded with a freedom of positional relationship twisted into a helical structure. It is.

1) PI polyamide (8 base recognition)
A PI polyamide of AcImPyβImImPyPyγImImPyβImImPyβDp (Chemical Formula 1) was designed so as to recognize the base sequence (8 base sequences) shown in the red part of FIG. The chemical formula of PI polyamide _{C 85 H 104 N 37 O 17} +, the molecular weight of 1915.97.
2) PI polyamide (12 base recognition)
PI polyamide of AcImPyPyβImPyβImImPyPyγImImPyβImImβImImImPyβDp (Chemical Formula 2) was designed so as to recognize the base sequence (12 base sequences) shown in the red part of FIG. The chemical formula of this PI polyamide was C ₁₂₃ H ₁₄₆ N ₅₅ O ₂₅ ⁺ and the molecular weight was 279.83.
As a PI polyamide that recognizes a base sequence of 8 bases or more, a complex with a tandem hairpin structure PI polyamide or DNA is theoretically possible. In, no one that recognizes a base sequence of 8 bases or more was known. Furthermore, these PI polyamides were novel ones that can recognize base sequences rich in GC sequences.
3) PI polyamide (7 base recognition)
A PI polyamide of AcPyPyβPyPyImγPyPyPyβImImβDp (Chemical Formula 3) (Chemical Formula 3) was designed so as to recognize the base sequence (7 base sequence) shown in the red part of FIG. The chemical formula of this PI polyamide was 1667.77C ₇₇ H ₉₆ N ₂₉ O ₁₅ ⁺ , and the molecular weight was 1667.77.

2. Synthesis of PI polyamide <Synthesis of pyrrole imidazole polyamide using HCTU>
Using HCTU (Peptide Institute, Inc.) as a condensation activator, the above 1. PI polyamides (3 types) designed in the above were synthesized.
1. Preparation of Reagent 1) Monomers FmocPyCOOH (Wako, 36 mg), FmocImCOOH (Wako, 77 mg), Fmoc-γ-Abu-OH (Nova Biochem, 34 mg) and Fmoc-β-Ala-OH (Nova Biochem, 32 mg), respectively The required coupling amount was prepared for each PI polyamide to be synthesized, and 4 equivalents of FmocImCOOH and 2 equivalents of the other were weighed with respect to the resin, and transferred to a 1.5 mL Eppendorf tube. Further, 88 mg of HCTU was added to the FmocImCOOH tube, and 44 mg was added to the other tubes. In addition, 500 μL of NMP (manufactured by Nacalai tesque) was added to a tube of FmocImCOOH, 250 μL was added to the other tubes, and vortexed for 1 hour to completely dissolve.
2) Reagent for synthesis The reagents listed in Table 1 were prepared and used for synthesis by a synthesizer.

3. Preparation of Resin 40 mg (0.04 mmol) of Fmoc-β-Ala-Wang-Resin (manufactured by Peptide Institute) was taken in Small Libra Tube (manufactured by HiPep Laboratories) and set in a peptide synthesizer. To this was added 1 mL of NMP and allowed to swell for 20 minutes.

4). Peptide synthesis (automatic)
Above 1. The DIEA prepared in (1) was installed as a condensation activator in a synthesizer (PSSM-8; manufactured by Shimadzu Corporation). Moreover, the tube containing each monomer prepared previously was arrange | positioned in the rack in a synthesis machine in order from C terminal. Set the synthesis program of PSSM-8, synthesizer is _started, H 2 _{NImPyβImImPyPyγImImPyβImImPyβ-Resin,} to H 2 NImPyPyβImPyβImImPyPyγImImPyβImImβImImImPyβ-Resin or _H 2 _{NPyPyβPyPyImγPyPyPyβImImβ-Resin,} repeating the reaction cycle of the following (1) to (4) The automatic synthesis was performed.

Reaction cycle (1) The coupling treatment was performed in NMP for 30 minutes using the above activator.
(2) Resin washing with 1 mL NMP was repeated 5 times to remove excess monomer and activator.
(3) 1 mL of Fmoc deprotection solution (30% Piperidine / NMP) was added and reacted for 3 minutes. After removing the solution, the same cycle was repeated again.
(4) To remove the Fmoc deprotection solution, washing of the resin with 1 mL of NMP was repeated 5 times to return to (1). This cycle was repeated until the desired product was obtained.

5. Purification The resin was removed from the synthesizer, washed and dried, and then transferred to an eppendorf tube with a screw cap. To this, 500 μL of N, N-dimethylpropanamine (manufactured by Nacalai tesque, 2 mL) was added and heated at 55 ° C. overnight with a heat block to cut out the polyamide from the resin. The reaction solution was transferred to a Libra Tube, the resin was removed by filtration, and the remaining reaction solution adhering to the resin was collected with 1 mL of NMP and 1 mL of methanol.
After the solvent was distilled off, purification was performed by HPLC. The HPLC after preparative purification is shown in FIG. 3 (0.1% AcOH: CH ₃ CN = 100: 0 to 0: 100, ₃₀ min). After preparative purification, it is freeze-dried and the above 1. PI polyamides (3 types) designed in the above were obtained.

[Test Example 1]
<Confirmation of sequence specificity of PI polyamide>
The sequence specificity of the PI polyamide (chemical formula 3) synthesized in Example 1 for the AP2 binding site of human ABCA1 was confirmed by gel shift assay.
1. Sample Preparation and Test 1) FITC conjugated oligo DNA (SEQ ID NO: 1) containing an AP2 binding site to which PI polyamide binds and its complementary strand were synthesized by consignment (manufactured by Invitrogen).
That is, a mixture of the complementary strand and FITC conjugated oligo DNA so as to have the composition shown in Table 2 was incubated at 95 ° C. for 5 minutes. Subsequently, double-stranded DNA was prepared by slowly lowering the temperature to room temperature.
2) The double-stranded DNA of 1) above was directly used in the gel shift assay (FIGS. 4, 1.).
3) A double-stranded DNA of 1) above and an equal amount (equal mole) of PI polyamide (Chemical Formula 3) mixed at the composition shown in Table 3 were incubated at room temperature for 1 hour (FIG. 4, 2.).
4) As a specific competitor, 10-fold amount of non-labeled oligo DNA (oligo DNA without FITC modification) was added to FITC conjugated oligo DNA, and double-stranded DNA and PI polyamide (Formula 3) are shown in Table 4. The mixture of the composition described was incubated at room temperature for 1 hour (FIGS. 4, 3).
5) FITC conjugated oligo DNA and mismatched PI polyamide (AcPyPyβPyImPyγPyPyPyβImPyβDp, chemical formula: C ₈₉ H ₁₀₈ N ₃₃ O ₁₇ , molecular weight: 1666.6) described in Table 5 The mixture was incubated at room temperature for 1 hour (FIGS. 4 and 4).
6) Each of the above 2) to 5) was preliminarily electrophoresed in 0.5 × TBE buffer (composition: 44.5 mM Tris, 44.5 mM boric acid, 1 mM EDTA (pH 8.0) for 20 minutes at 20 V) The solution was applied to% polyacrylamide gel and electrophoresed at 350V for 3 hours.
7) The gel after migration was photographed with LAS3000 (manufactured by FUJIFILM). The results are shown in FIG.

　

2. Results FIGS. 1, the mobility is changed by binding of double-stranded DNA to PI polyamide. It was confirmed to shift to the position.
Also, FIGS. , It was confirmed that the binding to FITC conjugated oligo DNA decreased because PI polyamide was also bound to non-labeled oligo DNA. From this result, it was shown that double-stranded DNA and PI polyamide were bound in a sequence-specific manner.
Furthermore, FIGS. 2 confirmed that the double-stranded DNA and the mismatched PI polyamide do not bind to each other, and that the binding does not occur if the sequences do not match.
Similarly, in the PI polyamide (Chemical Formula 1, Chemical Formula 2) synthesized in Example 1, the sequence specificity for the AP2 binding site of human ABCA1 was confirmed.

[Test Example 2]
<Effect of PI polyamide on ABCA1 gene expression>
1. Sample 1) PI polyamide The PI polyamide synthesized in Example 1 (chemical formulas 1 to 3) was used.
2) Cells: THP-1 cells (JCRB0112) (Human (peripheral blood, liquefied leukemia, lymphocyte-like) (RP-GBS) containing 10% of FRP in the form of 10% FIT-it BS from Human Science) Cultured on a 6-well plate. The cells were cultured at phorbol 12-myristate 13-acetate (PMA) 3.2 × 10 ⁻⁷ M (final concentration) for 72 hours to differentiate into THP-1 macrophages. All cells were cultured at 37 ° C. with 5% CO ₂ gas.

2. Confirmation of ABCA1 gene expression THP-1 macrophages of 1.2) above were treated with PI polyamide 1 uM, doxazosin (Doxazosin, Sigma (D9815)) 10 uM (positive control) or Go6983 (Sigma (G1918)) 10 nM (positive control). And 10% FBS-containing RPMI-1640 medium each supplemented with a 6-well plate for 16 hours.
After culturing for 16 hours, the medium was discarded, the cells were collected with TRIzol, and total RNA was purified (manufactured by Invitrogen). The purified total RNA was reverse-transcribed with a high capacity cDNA reverse transcription kit (manufactured by applied biosystems).
Then, real-time PCR was performed using Primer (SEQ ID NO: 2 and 3) synthesized by consignment (invitrogen) and using Power SYBR Green PCR Master Mix (manufactured by Applied Biosystems (43667659)). In addition, Taqman probe method (human 18S rRNA: applied biosystems (product number 4352930E) TaqMan Universal Master Mix II, with UNG (Appli Biosystems made by AIS, 444I) Time PCR was performed.
The obtained data was corrected with the results of 18S rRNA and shown in FIGS.

3. Results As shown in FIG. 5, even when PI polyamide (Chemical Formula 3) (FIG. 5, PyIm) was used, doxazosin (FIG. 5, Doxazosin) or Go6983 despite the low concentration of PI polyamide of 1 μM. It was confirmed that the expression of ABCA1 gene (mRNA) was significantly increased as compared with the control as in the case of using.
In addition, as shown in FIG. 6, the concentration of each PI polyamide is not affected by using any of PI polyamide (chemical formulas 1 to 3) (FIG. 6, 7mer: chemical formula 3, 8mer: chemical formula 1, 12mer: chemical formula 2). However, it was confirmed that the expression of ABCA1 gene (mRNA) increased despite the low concentration of 1 μM.

[Test Example 3]
<Effect of PI polyamide on extracellular lipid withdrawal in the presence of Apo-AI>
1. Sample PI polyamide (chemical formulas 1 to 3) synthesized in Example 1 and THP-1 macrophages obtained in the same manner as in Test Example 2 were used.

2. Confirmation of extracellular lipid withdrawal in the presence of Apo-AI THP-1 macrophages were added with 0.1% BSA containing PI polyamide 1 uM, doxazosin 10 uM (positive control) or Go6983 10 nM (positive control), apo -AI was cultured in a 6-well plate for 16 hours in RPMI-1640 medium containing 10 ug / ml. Apo AI was obtained by collecting HDL fraction from human plasma by ultracentrifugation, degreased with ethanol: ether (3: 2), and then obtained by Sephacryl S-200 gel chromatography.
After 16 hours of culture, the medium of each well was collected, and the lipid contained therein was extracted with a chloroform: methanol (2: 1) mixture, dried under reduced pressure, and then dissolved with isopropanol. Free cholesterol and phospholipid contained in the extracted lipids were measured by free cholesterol E-Test Wako (Wako chemicals (435-35801)) or phospholipid C-Test Wako (Wako chemicals (433-36201)), respectively. .
The cells were lysed with a RIPA buffer (manufactured by Nacalai tesque (08714)), and the amount of protein was measured with a BCA kit (manufactured by Thermo Scientific (# 23227)). The measured amounts of free cholesterol and phospholipid were corrected by the amount of protein and are shown in FIGS.

3. Results As shown in FIG. 7 (FC: Free cholesterol, PL: Phospholipid), even when PI polyamide (chemical formula 3) is used, the concentration of PI polyamide is as low as 1 μM. Regardless, as with doxazosin (FIG. 7, Doxazosin) or Go6983, the amount of free cholesterol and phospholipids was higher compared to controls. Therefore, from this result, it was confirmed that PI polyamide (FIG. 7, PyIm) is useful for extracting lipid out of the cell.
Further, as shown in FIG. 8, the concentration of each PI polyamide is not affected by using any of PI polyamide (chemical formulas 1 to 3) (FIG. 8, 7mer: chemical formula 3, 8mer: chemical formula 1, 12mer: chemical formula 2). Despite the low concentration of 1 μM, the amount of phospholipid was higher than the control. Therefore, from this result, it was confirmed that any of the PI polyamides (chemical formulas 1 to 3) is useful for extracting lipids from the cell.

[Test Example 4]
<Effect of PI polyamide on expression of ABCA1 protein>
1. Sample PI polyamide (chemical formula 3) synthesized in Example 1 and THP-1 macrophages obtained in the same manner as in Test Example 2 were used.

2. Confirmation of ABCA1 protein expression THP-1 macrophages were cultured in a 6-well plate in 10% FBS-containing RPMI-1640 medium supplemented with PI polyamide 1 uM, doxazosin 10 uM (positive control) or Go6983 10 nM (positive control), respectively, for 24 hours.
After 24 hours of culturing, the medium was discarded, the cells were washed with PBS, and then lysed with a RIPA buffer (manufactured by nacalai tesque (08714)) containing 1% Triton X-100, 2 mM 2-mercaptoethanol. Perform SDS-PAGE with 4-20% gradient gel, transfer to PVDF membrane, express with ECL Plus reagent (GE Healthcare (RPN2132)) using anti-ABCA1 antibody (Santa Cruz Biotechnology (SC-5491)) The ABCA1 protein was detected.

3. Results As shown in FIG. 9, even when PI polyamide (Chemical Formula 3) (FIG. 9, PyIm) was used, doxazosin (FIG. 9, Doxazosin) or Go6983 despite the low concentration of PI polyamide of 1 μM. It was confirmed that the expression of ABCA1 protein was increased as compared with the control as in the case of using.

<Anti-sclerosis agent>
An anti-arteriosclerotic agent was produced using one or more PI polyamides synthesized in Example 1 as active ingredients.

[Test Example 5]
<Effect of PI polyamide in blood HDL increasing action in vivo>
1. Confirmation of the effect of PI polyamide in vitro It binds to the site centering on the AP2 binding site of the mouse ABCA1 gene promoter region (recognition site: base sequence shown in the red part of Fig. 2 (4) (7 base sequence)) PI polyamide (AcPyPyImβImImγPyPyPyPyPyPyPyPyIβDp (chemical formula 4), hereinafter referred to as mABC1), chemical formula: C ₇₆ H ₉₅ N ₃₀ O ₁₅ ⁺ , molecular weight: 1666.93) Was used.
In the same manner as in Test Example 1, the sequence specificity of mABC1 for the AP2 binding site of mouse ABCA1 was confirmed by gel shift assay. As a result, as shown in FIG. 10, it was confirmed that mABC1 has sequence specificity with respect to the sequence specificity of mouse ABCA1 for the AP2 binding site.
Next, the expression of the mouse ABCA1 gene was confirmed in the same manner as in Test Example 2 using mouse macrophage cells (RAW 264cell) obtained from RIKEN BioResource Center, and Apo-AI was tested in the same manner as in Test Example 3. The effect on the extraction of lipids in the presence of cells in the presence was confirmed.
As a result, as shown in FIG. 11, mABC1 (FIG. 11, PyIm) significantly increased the expression of the ABCA1 gene in RAW264cel in the same manner as doxazosin (FIG. 11, Dox) as a positive control. Further, it was confirmed that mABC1 (FIGS. 12, 13 and PyIm) significantly increases the extraction of both free cholesterol (FIG. 12) and phospholipid (FIG. 13) in the extraction of lipid to the outside of cells.

2. Confirmation of the effect of PI polyamide in vivo C57BL / 6J mice 9-week-old male (Oriental yeast stock) reared with feed MF (Oriental Yeast Co., Ltd.) and water for mouse rat hamster at room temperature 22 ± 5 ° C. Company).
The mice were divided into a control group for administering mismatched PI polyamide (same as that used in Test Examples 1 and 5) and an experiment (PI polyamide (hereinafter referred to as mABC1) administration) group (n = 5), each PI polyamide was dissolved in 0.1% acetic acid, and 1 mg / kg BW was administered 4 times every other day from the tail vein. On day 10 from the start of administration, blood was collected from the heart and the liver was removed from each mouse under anesthesia.
Plasma was separated from this blood and subjected to agarose gel electrophoresis. As a result, as shown in FIG. 14, the HDL fraction increased in the mABC1 administration group (FIG. 14, P-2 to P-5) compared to the control group (FIG. 14, C-3 to C-6). Was recognized. Further, when detailed changes were examined by HPLC, it was confirmed that large HDL and very large HDL were increased in the mABC1 administration group.
Furthermore, detailed lipoprotein analysis in plasma was performed by HPLC, and total RNA was collected from leukocytes and liver tissues in the blood of each mouse, and the expression of ABCA1 gene (mRNA) was examined. As a result, it was confirmed that the expression of ABCA1 gene (mRNA) in leukocytes was higher in the PI polyamide administration group although there was no significant difference. It was also confirmed that the expression level of ABCA1 gene (mRNA) in the liver was significantly increased compared to the control group.

In these results, PI polyamide that binds to the site centering on the AP2 binding site of the mouse ABCA1 gene promoter region increases blood HDL and increases the expression level of the liver ABCA1 gene (mRNA) in vivo. It was confirmed to have an action. Therefore, based on these results, it was predicted that PI polyamide (chemical formulas 1 to 3) that binds to a site centered on the AP2 binding site of the human ABCA1 gene promoter region prepared in the present invention also has the same action.

The PI polyamide provided by the present invention can be used as an active ingredient of a blood HDL increasing agent and an anti-arteriosclerotic agent that are stable and effective. Further, provision of a drug containing PI polyamide as an active ingredient makes it possible to synthesize PI polyamide that is more useful in the prevention and treatment of arteriosclerotic diseases.

Claims (3)

1. A blood HDL increasing agent comprising one or more PI polyamides of chemical formulas 1 to 3 as an active ingredient.
   [Chemical Formula 1]
   

   

   
   
   [Chemical formula 2]
   

   

   
   [Chemical formula 3]
   

   

   
   
2. An anti-arteriosclerotic agent comprising one or more PI polyamides of chemical formulas 1 to 3 as an active ingredient.
3. A PI polyamide according to Chemical Formula 1 or 2.

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