JP2023087493A - Composition for regulating gene expression - Google Patents

Abstract

To provide a new composition for inhibiting inflammation reaction of a vascular endothelial cell.SOLUTION: A composition comprises a compound represented by the following formula or a salt thereof. [R1 and R2 each represent NH or the like, R3 represents a halogen atom; R4's independently represent H or the like, R5 represents S or O, R6 represents a carboxyl group, R7 represents NH2 or the like, and n represents 1-11].SELECTED DRAWING: None

Description

The present disclosure relates to compositions for modulating gene expression. More specifically, it relates to compositions for modulating specific gene expression in vascular endothelial cells.

Inflammatory reaction is a protective and adaptive reaction that originally acts as a defense mechanism when the body receives external and internal damage. However, when external and internal damage persists over a long period of time, a low-level inflammatory response called chronic inflammation persists and prolongs, resulting in tissue function modulation/disorder and even fibrosis. It is thought that it causes irreversible organ failure due to degeneration. In recent years, much attention has been paid to chronic inflammation as an underlying pathophysiology common to various diseases, especially chronic diseases (eg, Non-Patent Document 7).

The infectious disease (COVID-19) caused by the new coronavirus (SARS-CoV2), which is said to have started infecting humans since the end of 2019, is raging all over the world. Furthermore, even after recovery from COVID-19, many patients suffer from aftereffects such as general malaise, loss of taste and smell, and hair loss. In fact, among the COVID-19 patients who were forced to be hospitalized in Non-Patent Documents 1 and 2, nearly 30% of the patients were re-hospitalized in the latter half of the year, and about 12% of the patients died, or all It has been reported that as many as 33% of COVID-19 patients suffer from neurological or mental disorders after recovery, and the aftereffects of corona are becoming a very big social problem, but currently they are being treated. The problem is that there is no cure.

The novel coronavirus that causes COVID-19 has been suggested to infect cells that express angiotensin-converting enzyme 2 (ACE2) on their surface. Expression of ACE2 has been confirmed not only in lung alveolar epithelial cells but also in vascular endothelial cells and vascular smooth muscle cells throughout the body, and recent findings have revealed that it inhibits the function of vascular endothelial cells. (Non-Patent Document 3), and vascular inflammation is thought to be one of the reasons why the symptoms of COVID-19 spread not only to the lungs but to the whole body (Non-Patent Document 4). And even if one recovers from COVID-19, the damage to the blood vessels throughout the body remains, causing various symptoms afterwards. is also considered.

In fact, PAI-1 inhibitors for lung injury/respiratory failure associated with COVID-19 have progressed to the stage of aiming for early approval as soon as phase II clinical trials are completed (Non-Patent Document 8). However, this drug is a drug that inhibits PAI-1, and there is also the problem that it is left to the patient's own natural healing power for lung damage and respiratory failure caused before drug administration. there is

Furthermore, as in Non-Patent Document 5, it has been reported that vascular endothelial cells receive signals from inflammatory cytokines and cause premature senescence (premature aging), and inflammation causes vascular aging. It is clear that For example, it has been shown that TNFα stimulation promotes the expression of E-selectin, a type of adhesion molecule, and traps immune cells in vascular endothelial cells, thereby inducing thrombi (Non-Patent Document 5). ). It has also been reported that the levels of various inflammatory cytokines in serum increase with actual human aging (Non-Patent Document 6). presumably related.

https://www.medrxiv.org/content/10.1101/2021.01.15.21249885v1 https://www.thelancet.com/journals/lanpsy/article/PIIS2215-0366(21)00084-5/fulltext https://www.ahajournals.org/doi/10.1161/CIRCRESAHA.121.318902 https://jglobal.jst.go.jp/detail?JGLOBAL_ID=202002275608240567 https://www.nature.com/articles/srep39501 https://link.springer.com/article/10.1007/s00005-015-0357-7 http://plaza.umin.ac.jp/manabe/project/inflammation/ https://bio.nikkeibp.co.jp/atcl/news/p1/21/09/13/08625/

Thus, inflammation, especially vascular inflammation, is considered to be a very important pathological condition that can affect the whole body. In fact, there are diseases that cause symptoms in various parts of the whole body due to the symptoms of vasculitis. Anti-inflammatory agents such as steroids and antibody drugs such as Actemra (anti-IL-6 antibody) and Humira (anti-TNFα antibody) have been used to suppress inflammation. However, there was still a need for new approaches with new active ingredients. Thus, the present disclosure provides 1) inflammatory cytokines (IL-6, IL-8) and/or 2) adhesion molecules (E-selectin, VCAM1, ICAM1), and 3) It is an object of the present invention to provide a composition for suppressing the expression of fibrinolysis inhibitory factor (PAI-1).

As a result of extensive studies by the inventors, when the low-molecular-weight compound AS1842856 was added to HUVEC (Human umbilical vein endothelial cells), which is a type of vascular endothelial cells, inflammatory cytokines IL-6 and IL -8 expression induction was found to be remarkably suppressed.

Furthermore, under the conditions described above, it was found that all mRNA levels of adhesion molecules E-selectin, VCAM1, ICAM1 and PAI-1 were significantly suppressed.

Furthermore, they found that the mRNA level of the fibrinolytic inhibitor PAI-1 was significantly suppressed under the conditions described above.

IL-6, IL-8 and PAI-1 are common genes expressed when various cells age, called SASP (Senescence Associated Secretory Phenotype), and are closely related to aging. Since AS1842856 significantly suppresses these mRNA levels, it was found that AS1842856 also suppresses senescence of vascular endothelial cells.

｛ただし、
Ｒ¹は、ＮＨ又はＣＨ₂であり、
Ｒ²は、Ｎ－Ｒ⁸又はＣＨ－Ｒ⁸（Ｒ⁸：Ｈ、又はＣ１～Ｃ４までのアルキル基）であり、
Ｒ³は、Ｆ、Ｃｌ、Ｂｒ、又はＩであり、
Ｒ⁴は、それぞれ独立して、Ｈ又はＣ１～Ｃ２までのアルキル基であり、
Ｒ⁵は、Ｓ又はＯであり、
Ｒ⁶は、カルボキシル基であり、
Ｒ⁷は、ＮＨ₂、又はＣ１～Ｃ２までのアルキル基であり、
ｎは、１－１１である。｝
（発明２）
発明１の組成物であって、前記化合物が、以下の式で表される、組成物。

｛ただし、
Ｒ¹は、ＮＨであり、
Ｒ²は、Ｎ－Ｒ⁸（Ｒ⁸：Ｃ１～Ｃ３までのアルキル基）であり、
Ｒ³は、Ｆ、Ｃｌ、Ｂｒ、又はＩであり、
Ｒ⁴は、それぞれ独立して、Ｈ又はＣＨ₃であり、
Ｒ⁵は、Ｏであり、
Ｒ⁶は、カルボキシル基であり、
Ｒ⁷は、ＮＨ₂であり、
ｎは、１－１１である。｝
（発明３）
発明１又は２の組成物であって、前記炎症反応に関する遺伝子は、ＩＬ－６、ＩＬ－８、Ｅ－ｓｅｌｅｃｔｉｎ、ＶＣＡＭ１、ＩＣＡＭ１、及び線溶阻害因子（ＰＡＩ－１）から選択される１種以上である、組成物。
（発明４）
発明１～３いずれか１つに記載の組成物であって、前記炎症反応に関する遺伝子が、炎症性サイトカインＩＬ－６及び／又はＩＬ－８である、組成物。
（発明５）
発明１～３いずれか１つに記載の組成物であって、前記炎症反応に関する遺伝子が、接着分子Ｅ－ｓｅｌｅｃｔｉｎ、ＶＣＡＭ１、及びＩＣＡＭ１から選択される１種以上である、組成物。
（発明６）
発明１～３いずれか１つに記載の組成物であって、前記炎症反応に関する遺伝子が、線溶阻害因子（ＰＡＩ－１）である、組成物。
（発明７）
発明１～３いずれか１つに記載の組成物であって、炎症による血管内皮細胞の老化を抑制するための組成物。
（発明８）
発明１～３いずれか１つに記載の組成物であって、血管炎疾患の治療、又は、進行を遅らせるための組成物。 The invention of the present disclosure has been completed based on the above findings, and includes the following inventions in one aspect.
(Invention 1)
A composition for suppressing the expression of genes related to inflammatory response in vascular endothelial cells,
The composition comprises a compound represented by the following formula or a salt thereof.

{however,
^R1 is NH or _CH2 ;
R ² is N—R ⁸ or CH—R ⁸ (R ⁸ : H or a C1-C4 alkyl group);
^R3 is F, Cl, Br, or I;
each R ⁴ is independently H or a C1-C2 alkyl group;
^R5 is S or O;
^R6 is a carboxyl group,
R ⁷ is NH ₂ or a C1-C2 alkyl group;
n is 1-11. }
(Invention 2)
The composition of Invention 1, wherein the compound is represented by the following formula.

{however,
R ¹ is NH;
R ² is NR ⁸ (R ⁸ is an alkyl group of C1 to C3);
^R3 is F, Cl, Br, or I;
each ^R4 is independently H or _CH3 ;
^R5 is O;
^R6 is a carboxyl group,
^R7 is _NH2 ;
n is 1-11. }
(Invention 3)
The composition of Invention 1 or 2, wherein the gene related to inflammatory response is one selected from IL-6, IL-8, E-selectin, VCAM1, ICAM1, and inhibitor of fibrinolysis (PAI-1) A composition that is above.
(Invention 4)
4. The composition according to any one of Inventions 1 to 3, wherein the gene related to inflammatory response is inflammatory cytokine IL-6 and/or IL-8.
(Invention 5)
The composition according to any one of Inventions 1 to 3, wherein the gene related to inflammatory response is one or more selected from adhesion molecule E-selectin, VCAM1, and ICAM1.
(Invention 6)
The composition according to any one of Inventions 1 to 3, wherein the gene associated with inflammatory response is inhibitor of fibrinolysis (PAI-1).
(Invention 7)
A composition according to any one of Inventions 1 to 3, which is for suppressing senescence of vascular endothelial cells due to inflammation.
(Invention 8)
A composition according to any one of inventions 1 to 3, for treating or delaying the progression of vasculitic diseases.

In one aspect, the composition of the present disclosure contains an active ingredient represented by the chemical formula above. Thereby, genes related to inflammatory response in vascular endothelial cells (for example, 1) inflammatory cytokines (IL-6, IL-8) and/or 2) adhesion molecules (E-selectin, VCAM1, ICAM1) during inflammatory response, and/or 3) the expression of fibrinolytic inhibitor (PAI-1, etc.) can be suppressed.

Schematic diagrams of events occurring in vascular endothelial cells when inflammatory cytokines (eg, TNFα) are excessively released into blood vessels due to aging, bacterial/viral infection, or the like. Fig. 10 shows the result of examining the effect of AS1842856 on the enhancement of IL-6 and IL-8 expression by inflammatory cytokine TNFα stimulation in HUVEC using quantitative PCR (qPCR). Fig. 10 shows the result of examining the effect of AS1842856 on the enhancement of E-selectin, VCAM1 and ICAM1 expression by inflammatory cytokine TNFα stimulation in HUVEC using quantitative PCR (qPCR). Fig. 4 shows the result of examining the effect of AS1842856 on the enhancement of PAI-1 expression by inflammatory cytokine TNFα stimulation in HUVEC using quantitative PCR (qPCR). It is the result of examining the inflammatory cytokine TNFα stimulation in HUVEC and the effect of AS1842856 using comprehensive gene expression analysis. These are the results of examining the effect of insulin on the expression of IL-8 and the like in HUVEC. This is the result of examining the effect of HGF on the expression of IL-8 and the like in HUVEC.

Specific embodiments for carrying out the invention of the present disclosure will be described below. The following description is intended to facilitate understanding of the invention. it is not intended to limit the scope of the invention.

1. Definition The term "to inhibit the mRNA expression of XX whose expression is increased in vascular endothelial cells during an inflammatory response" described herein improves a specific blood vessel-related disease that occurs when the blood vessel is exposed to inflammation. include the purpose of Furthermore, the term is not limited to therapeutic purposes. For example, the term encompasses the purpose of preventing the development of a particular disease even if one does not have that disease.

Notably, the composition of the present disclosure in one embodiment can contribute not only to the treatment of vascular inflammation-related diseases, but also to the suppression of inflammatory responses in the blood vessels of aged people. For example, as described in Non-Patent Document 6 above, it has been revealed that the concentration of inflammatory cytokines in serum gradually increases with age. This result indicates that inflammatory damage accumulates in blood vessels with age, even if no particular disease condition develops. The composition of the present disclosure in one embodiment is also effective against inflammatory damage to blood vessels that accompanies such aging phenomenon, and so-called "anti-aging" effects are also expected.

Accordingly, in one embodiment, the compositions of the present disclosure may be formulated with suitable additional ingredients, depending on the mode of administration. For example, compositions of the present disclosure may include ingredients commonly included in pharmaceuticals. Examples of such ingredients include preservatives, preservatives, perfumes, pH adjusters, antioxidants, antifungal agents, and the like. In addition, since oral intake is also possible, processing such as tableting, encapsulation for oral use, and addition of artificial sweeteners may be performed.

The mode of administration is also not particularly limited, and examples thereof include oral administration and intravenous injection. A preferred form of administration is intravenous injection, taking into consideration the effect on vascular endothelial cells.

2. Active Ingredients In one embodiment, the present disclosure relates to compositions comprising an active ingredient. The active ingredient is a compound represented by the following structural formula or a salt thereof.

｛ただし、
Ｒ¹は、ＮＨ又はＣＨ₂であり、
Ｒ²は、Ｎ－Ｒ⁸又はＣＨ－Ｒ⁸（Ｒ⁸：Ｈ、又はＣ１～Ｃ４までのアルキル基）であり、
Ｒ³は、Ｆ、Ｃｌ、Ｂｒ、又はＩであり、
Ｒ⁴は、それぞれ独立して、Ｈ又はＣ１～Ｃ２までのアルキル基であり、
Ｒ⁵は、Ｓ又はＯであり、
Ｒ⁶は、カルボキシル基であり、
Ｒ⁷は、ＮＨ₂、又はＣ１～Ｃ２までのアルキル基であり、
ｎは、１－１１である。｝

{however,
^R1 is NH or _CH2 ;
R ² is N—R ⁸ or CH—R ⁸ (R ⁸ : H or a C1-C4 alkyl group);
^R3 is F, Cl, Br, or I;
each R ⁴ is independently H or a C1-C2 alkyl group;
^R5 is S or O;
^R6 is a carboxyl group,
R ⁷ is NH ₂ or a C1-C2 alkyl group;
n is 1-11. }

The salt form is not particularly limited, and may be sodium salt, potassium salt, or the like.

In a preferred embodiment, the compound may be a compound represented by the following formula or a salt thereof.

｛ただし、
Ｒ¹は、ＮＨであり、
Ｒ²は、Ｎ－Ｒ⁸（Ｒ⁸：Ｃ１～Ｃ３までのアルキル基）であり、
Ｒ³は、Ｆ、Ｃｌ、Ｂｒ、又はＩであり、
Ｒ⁴は、それぞれ独立して、Ｈ又はＣＨ₃であり、
Ｒ⁵は、Ｏであり、
Ｒ⁶は、カルボキシル基であり、
Ｒ⁷は、ＮＨ₂であり、
ｎは、１－１１である。｝

{however,
R ¹ is NH;
R ² is NR ⁸ (R ⁸ is an alkyl group of C1 to C3);
^R3 is F, Cl, Br, or I;
each ^R4 is independently H or _CH3 ;
^R5 is O;
^R6 is a carboxyl group,
^R7 is _NH2 ;
n is 1-11. }

In a most preferred embodiment, the compound may be a compound represented by the following formula (CAS No. 836620-48-5) or a salt thereof.

3. Other Components of Composition In one embodiment described above, the composition may optionally contain other components in addition to the compounds described above. For example, depending on the application, the composition may include one or more of the following ingredients.
pH adjuster (e.g., phosphate buffer, Tris buffer, etc.)
Inorganic salts (e.g. sodium chloride, potassium chloride, etc.)
Sugars (e.g. lactose, sucrose, etc.)
Excipients (e.g. water, purified water, alcohol, glycerin, lactose, starch, dextrin, sucrose, precipitated silica, etc.)

Also, the composition may be in solid or liquid form. In the case of a liquid, the solvent is not particularly limited, and may be water or an organic solvent.

4. Genes of Interest In one embodiment, the compositions of the present disclosure can suppress the expression of genes associated with inflammatory responses in a cell. For example, genes to be suppressed include the following genes.
(1) Inflammatory cytokines ⇒ for example, IL-6, IL-8
(2) Adhesion molecules ⇒ For example, E-selectin, VCAM1, ICAM1
(3) fibrinolytic inhibitor PAI-1

4-1. inflammatory cytokine
Inflammatory cytokines are involved as causative factors that cause various inflammatory symptoms in vivo. In one embodiment, the compositions of the present disclosure suppress expression of proinflammatory cytokine genes. Examples of inflammatory cytokine genes include IL-6, IL-8 and the like.

4-2. Adhesion molecules involved in inflammation include E-selectin, VCAM1 and ICAM1. Adhesion molecules involved in inflammation have the function of inducing excessive accumulation of immune cells and thrombus formation. Specifically, E-selectin plays an important role in the recruitment of leukocytes to the site of injury during inflammation. In addition, VCAM-1 is significantly enhanced on vascular endothelial cells within several hours by stimulation with inflammatory cytokines, active oxygen, etc., and adheres to lymphocytes and eosinophils in the circulating blood and tissues. and activation. ICAM1 increases in endothelial cells within several hours upon stimulation with inflammatory cytokines, active oxygen, etc., and participates in the adhesion and activation of inflammatory cells in circulating blood and tissues.

4-3. fibrinolytic inhibitor PAI-1
When PAI-1 expression increases locally in blood vessels due to inflammation, it not only increases the fibrin thrombus, but also inhibits the dissolution of the extracellular matrix to promote its deposition on the vascular wall, which is thought to advance arteriosclerotic lesions.

5. Tissues or cells of interest In one embodiment, the compositions of the present disclosure are used to target specific cells. Here, the specific cells are vascular endothelial cells.

6. EXAMPLES Below are further specific examples related to the above embodiments. Similar to the above embodiments, the following examples are provided for the purpose of promoting understanding of the invention and are not intended to limit the scope of the invention.

6-1. Culture of vascular endothelial cells Human umbilical vein endothelial cells (HUVEC; Takara, C-12208) were prepared. The cells were passaged and maintained using Endothelial Cell Growth Medium 2 Kit, Takara, C-22111.

HUVECs were seeded into each well of a 12-well plate (Corning, 3336) at 6,000 cells/cm ² and cultured overnight. The next day (16-24 hours after seeding), AS1842856 was added at multiple concentrations (10 nM, 100 nM, 1 μM) or DMSO used as its solvent was added as a control. Immediately thereafter, 10 ng/ml TNFα (PeproTech, AF-300-01A) or PBS(-) as a control was added and cultured for 1 day (about 24 hours). After culturing, total RNA was extracted from HUVEC using ReliaPrep RNA Miniprep system (Promega, Z6012). After extraction, cDNA synthesis (PrimeScript RT Master Mix; Takara, RR036A) was performed using 500 ng of RNA, and quantitative PCR (Thunderbird Sybr qPCR Mix; TOYOBO, QPS-201X5) was performed.

6-2. cDNA Synthesis A mixture for cDNA synthesis was prepared according to the following composition.
2 μl of 5x PrimeScript RT Master Mix (1x final concentration)
Total RNA 500ng
Adjusted to 10 μl in total of RNase free H ₂ O

The above mixture was processed using a Veriti 96 well Thermal Cycler manufactured by Applied Biosystems under the following conditions.
37°C 15 minutes ↓
85°C 5 seconds ↓
4°C ∞

The synthesized cDNA (10 µl) was diluted 10-fold with 90 µl of TE (10 mM Tris-HCl pH 8.0 + 1 mM EDTA pH 8.0). The dilutions were subjected to quantitative PCR.

6-3. More specifically, for analysis of gene expression in HUVEC by qPCR , the dilutions were mixed under the following conditions.
10 μl of 2x THUNDERBIRD Probe qPCR Mix
0.4 μl of 5 mM Forward Primer
0.4 μl of 5 mM Reverse Primer
_H2O 8.2 μl
cDNA (10-fold dilution) 1 μl

cDNA was amplified from the above mixture using Bio-Rad's CFX-Connect. The PCR cycle conditions were as follows.
1.95°C 1 minute (initial denaturation)
2.95°C 15 seconds (denaturation)
3.60°C 30 seconds (elongation)
(Repeat steps 2-3 40 times, detect fluorescence signal each time step 3 is completed)
4. Raise the temperature from 65°C to 95°C in increments of 0.5°C, hold the temperature for 5 seconds, and then detect the fluorescence signal.

The PCR product was detected in the above cycle, and the identity of the PCR product was confirmed by melting curve.

GAPDH ( Glyceraldehyde 3- phosphate dehydrogenase ) was used as an internal standard (a housekeeping gene expressing the same expression in all cells and conditions).
The primer sequences for detecting each gene were as follows.
GAPDH Forward primer: 5'-agccacatcgctcagacac-3'
GAPDH Reverse primer: 5'-gcctaatacgaccaaatcc-3'
IL-6 Forward primer: 5'-gatgagtacaaaagtcctgatccaa-3'
IL-6 Reverse primer: 5'-ctgcagccactggttctgt-3'
IL-8 Forward primer: 5'-agacagcagagcacacaagc-3'
IL-8 Reverse primer: 5'- atggttccttccggtggt-3'

All primers were purchased from Fasmac Co., Ltd. (reverse phase column purification grade).
The expression level of each gene indicates the standardized value obtained by dividing the expression level of each gene obtained by quantitative PCR by the expression level of GAPDH. Furthermore, the expression level under the condition that TNFα was added and AS1842856 was not added was standardized to be "1". It was confirmed by a melting curve that the PCR products amplified using the above primers were all single (that is, multiple types of sequences were not amplified with the same primers).

The results are shown in FIG. First, in the presence of DMSO, TNFα (10 ng/ml) treatment markedly enhanced the expression of inflammatory cytokine IL-6 and IL-8 mRNA compared to the control PBS(-). On the other hand, when AS1842856 was added, it was observed that the elevation of IL-6 and IL-8 mRNA expression due to TNFα (10 ng/ml) treatment was suppressed in a concentration-dependent manner. This result suggests the possibility that AS1842856 can suppress the expansion of inflammation caused by further secretion of inflammatory cytokines by vascular endothelial cells receiving inflammatory cytokines.

A test similar to that of Example 1 was performed. However, gene expression was detected using the following primers.
E-selectin Forward primer: 5' - accagcccaggttgaatg - 3'
E-selectin Reverse primer: 5' - ggttggacaaggctgtgc - 3'
VCAM1 Forward primer: 5' - tgcacagtgacttgtggacata - 3'
VCAM1 Reverse primer: 5'-gccaccactcatctcgattt-3'
ICAM1 Forward primer: 5'-ccttcctcaccgtgtactgg-3'
ICAM1 Reverse primer: 5'-agcgtagggtaaggttcttgcc-3'

Results for gene expression of adhesion molecules are shown in FIG. In the same manner as in Example 1 above, when TNFα (10 ng/ml) treatment was first performed in the presence of DMSO as compared with PBS (-) as a control, the expression of the mRNAs of the adhesion molecules E-selectin, VCAM1 and ICAM1 increased. markedly increased. On the other hand, when AS1842856 was added, it was observed that the increase in E-selectin, VCAM1 and ICAM1 mRNA expression due to TNFα (10 ng/ml) treatment was suppressed in a concentration-dependent manner. This result suggests the possibility that AS1842856 can suppress the expression of adhesion molecules and the induction of excessive accumulation and thrombus formation of immune cells when vascular endothelial cells receive inflammatory cytokines.

A test similar to that of Example 1 was performed. However, gene expression was detected using the following primers.
PAI-1 Forward primer: 5'-ccagctgacaacaggaggag-3'
PAI-1 Reverse primer: 5'-cccatgagctccttgtacagat-3'

Next, FIG. 4 shows the results regarding the gene expression of the fibrinolytic inhibitor PAI-1. In the same manner as above, first, in the presence of DMSO, TNFα (10 ng/ml) treatment markedly enhanced the expression of PAI-1 mRNA compared to the control PBS(-). On the other hand, when AS1842856 was added, it was observed that the increase in PAI-1 mRNA expression due to TNFα (10 ng/ml) treatment was suppressed in a concentration-dependent manner of AS1842856. This result suggests the possibility that AS1842856 can suppress the inhibition of thrombus dissolution by the expression of fibrinolysis-inhibiting factors when vascular endothelial cells receive inflammatory cytokines.

As described above, it was revealed that AS1842856 works to alleviate the phenotype of the inflammatory cytokine TNFα stimulation. Therefore, a comprehensive gene expression analysis was carried out to investigate what kinds of functions of the gene group other than the above genes are regulated (Fig. 5). For the comprehensive gene expression analysis, ThermoFisher's Ion Proton ^™ was used, and a technique called AmpliSeq was used to perform the expression analysis of approximately 20,000 genes. The sample used for the expression analysis was the RNA collected in the above experiment (simultaneous treatment in FIG. 2), and all experimental operations were performed according to the manual. The expression level of each gene is corrected by RPM ( Reads Per Million reads). First, genes whose expression level is increased by 2-fold or more by TNFα (10 ng / ml) stimulation are picked up. GO analysis (Gene Ontology analysis) was used to examine what functions these genes play. The public website "https://go.princeton.edu/cgi-bin/GOTermFinder" was used for GO analysis.
The results are shown in FIG. Interestingly, among the genes whose expression is increased by TNFα stimulation, most of the genes that are reduced to 1/2 or less by treatment with AS1842856 are particularly enriched, and most of them are related to cytokine signaling. It became clear that it was This result means that AS1842856 suppresses the expression of not only specific genes such as IL-6, but also many genes related to cytokine signals whose expression is elevated downstream of TNFα stimulation. It has been revealed that AS1842856 suppresses cytokine signals downstream of TNFα stimulation and acts to suppress excessive inflammatory reactions.

Next, tests similar to those of Examples 1 and 2 were performed. However, in addition to a pattern in which AS1842856 (1 μM) was administered, a pattern in which only DMSO was administered as a control, and a pattern in which insulin (10 μg/mL) was administered, samples were added.

The results are shown in FIG. The same results as in Example 1 were obtained in the pattern of administration of AS1842856 and the pattern of administration of only DMSO as a control. On the other hand, the insulin administration pattern showed a tendency similar to the DMSO administration pattern.

Tests similar to Examples 1 and 2 were performed. However, in addition to a pattern of administration of AS1842856 (1 μM) and a pattern of administration of only DMSO as a control, samples of a pattern of administration of PBS and a pattern of administration of HGF (10 ng/mL) were added.

The results are shown in FIG. The same results as in Example 1 were obtained in the pattern of administration of AS1842856 and the pattern of administration of only DMSO as a control. On the other hand, the pattern of administering PBS and the pattern of administering HGF showed a tendency similar to the pattern of administering only DMSO.

AS1842856 is known to have a transcription factor FoxO1 inhibitory effect. In addition, it is known that the transcription factor FoxO1 is involved downstream of the insulin signal and the HGF signal, and that the signal from these ligands inactivates the transcription factor FoxO1.

Based on these findings, referring to FIGS. 6 and 7, administration of insulin and HGF does not affect the expression of the above-described inflammatory cytokines, adhesion molecules, and fibrinolytic inhibitory factors, or the effect is remarkably small. This suggests that regulation of the transcriptional activity of the transcription factor FoxO1 per se does not significantly affect the expression of these genes. The results of Examples 5 and 6 show that AS1842856 affects the expression of inflammatory cytokine genes and anti-inflammatory cytokine genes by a novel mechanism different from the inhibitory action of transcription factor FoxO1. It suggests.

Specific embodiments have been described above. The above embodiments are only specific examples, and the present invention is not limited to the above embodiments. For example, technical features disclosed in one of the above embodiments may be applied to other embodiments. Also, unless otherwise stated, for a particular method, some steps may be interchanged with other steps, and additional steps may be added between two particular steps. The scope of the invention is defined by the claims.

Claims (8)

A composition for suppressing the expression of genes related to inflammatory response in vascular endothelial cells,
The composition comprises a compound represented by the following formula or a salt thereof.

{however,
^R1 is NH or _CH2 ;
R ² is NR ⁸ or CH-R ⁸ (R ⁸ : H, C1-C4 alkyl group);
^R3 is F, Cl, Br, or I;
each R ⁴ is independently H or a C1-C2 alkyl group;
^R5 is S or O;
^R6 is a carboxyl group,
R ⁷ is NH ₂ or a C1-C2 alkyl group;
n is 1-11. } 2. The composition of claim 1, wherein said compound has the formula:

{however,
R ¹ is NH;
R ² is NR ⁸ (R ⁸ is an alkyl group of C1 to C3);
^R3 is F, Cl, Br, or I;
each ^R4 is independently H or _CH3 ;
^R5 is O;
^R6 is a carboxyl group,
^R7 is _NH2 ;
n is 1-11. } 3. The composition of claim 1 or 2, wherein the gene associated with inflammatory response is selected from IL-6, IL-8, E-selectin, VCAM1, ICAM1, and inhibitor of fibrinolysis (PAI-1). A composition that is more than a seed. 4. The composition according to any one of claims 1 to 3, wherein said gene associated with inflammatory response is the inflammatory cytokines IL-6 and/or IL-8. 4. The composition according to any one of claims 1 to 3, wherein the gene related to inflammatory response is one or more selected from adhesion molecule E-selectin, VCAM1, and ICAM1. 4. The composition according to any one of claims 1 to 3, wherein the gene associated with inflammatory response is inhibitor of fibrinolysis (PAI-1). 4. The composition according to any one of claims 1 to 3, for suppressing senescence of vascular endothelial cells due to inflammation. A composition according to any one of claims 1 to 3 for treating or slowing the progression of vasculitic diseases.

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