JP2014040393A - Gene expression regulating agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new use of 2-aminoethanesulfonic acid.SOLUTION: An agent of regulating expression of a gene described in Table 1 and/or a homolog gene thereof, the agent containing 2-aminoethanesulfonic acid as an active ingredient.

Description

  The present invention relates to a new use of 2-aminoethanesulfonic acid.

  Various diseases such as asthma, cancer, heart disease, aneurysm, autoimmune diseases and viral infections have various symptoms, but are caused by abnormal expression (overexpression or underexpression) of one or several proteins Many diseases have been suggested. In general, the expression of these proteins is controlled through the regulation of mRNA expression by various transcription activators and transcription repressors. Therefore, it is thought that by regulating the expression of mRNA, the expression of the protein can be controlled, leading to improvement of diseases and symptoms.

For example, as an example of controlling the transcription factor itself, production of IL-1β, TNF-α, etc. that are excessively produced by controlling the activity of NF-κB is controlled, leading to treatment of psoriasis and atopic dermatitis ( Patent Document 1), control over the production of IL-6, TGF-β, etc., which are overproduced by controlling the activity of AP-1, restenosis, endometrial hyperplasia, benign prostatic hypertrophy, proliferative vitreous retina It is reported to be useful for treatment and improvement of diseases and symptoms such as symptom, pulmonary fibrosis, dry eye syndrome, and Parkinson's disease (Patent Document 2).
An example of a substance that regulates the activity of a transcription factor is a PPARγ agonist, which is a therapeutic drug for metabolic diseases, and the action mechanism of this drug is known to promote adipocyte differentiation. In addition, it has been reported that mechanisms such as transcription enhancement of adiponectin and transcriptional inhibition of TNF-α are also important (Non-patent Document 1). Furthermore, it is also known that, in the activated vitamin D3 preparation, the action of suppressing the overexpression of M-CSF and RANKL is important for the treatment of osteoporosis in addition to the regulation of calcium metabolism (Non-patent Documents 2 and 3).
In non-clinical situations, control of gene expression has been applied to various studies and tests as a useful reagent (Non-patent Document 4).
From these examples, the control of gene expression that shows abnormal expression in diseases and symptoms is useful for their treatment, alleviation, etc., and it is also useful for nonclinical studies and research to elucidate the onset mechanism and analyze disease states It is thought that.

Various genes such as cytokines, adhesion molecules, and intracellular signaling molecules are assumed as genes that are suggested to be associated with diseases and symptoms. For example, IL-1β has been reported to have enhanced gene expression in rheumatoid arthritis and symptom improvement by a receptor antagonist, and its expression control is considered useful for the treatment of rheumatoid arthritis (Non-patent Document 5). Moreover, the correlation between the expression of TREM1 and the severity of Behcet's disease has been reported, and appropriate regulation thereof is considered useful for treatment (Non-patent Document 6).
As described above, there are many known diseases and symptoms that are considered to be caused by abnormal expression of genes and proteins. However, among the genes expected to lead to treatment and improvement of such diseases and symptoms, only a part of the regulatory substances have been reported.

  2-Aminoethanesulfonic acid is a sulfur-containing amino acid that is abundant in fish and shellfish, and is present in the heart muscle, muscle, spleen, brain, lung, bone marrow, etc. in humans, and is used for medical drugs, OTC drugs, special-purpose foods, etc. Widely used in foods. Further, genes whose expression varies depending on 2-aminoethanesulfonic acid include NOS1, NOS2 (Non-patent Document 7), MMP9, FN1 (Non-patent Document 8), OLR1, ICAM1 (Non-patent Document 9), CTGF (non-patent document 7). Patent Document 10), CYP7A1 (Non-Patent Document 11), and the like are known.

Japanese Patent No. 4346233 Japanese Patent No. 4836961

Br J Pharmacol. 2008. 153: 636. Biochem Biophys Res Commun. 2002. 297: 1211. Journal of Cellular Biochemistry 2008. 105: 1289. Curr Med Chem. 2003. 10: 245. Ann Rheum Dis. 2004. 63: 1062. Inflamm Bowel Dis. 2011. 17: 2130. Exp Neurol. 2012. 233: 154. Biochem Pharmacol. 2011. 82: 1209. Ren Fail. 2008. 30: 763. Amino Acids. 2007. 32: 425. Amino Acids. 2006. 30:43.

  An object of this invention is to provide a novel gene expression regulator.

  In the present invention, there are provided an expression regulator of the genes listed in Table 1 and / or homologous genes thereof, and a preparation which can be used for prevention, diagnosis and treatment of diseases and symptoms associated with abnormal expression thereof. Although the genes listed in Table 1 and / or their homologous genes are presumed to be involved in various diseases and symptoms, they are intended for prevention, treatment, diagnosis, etc. containing substances that regulate their expression and active ingredients thereof Drugs are not known. In order to achieve the above object, the present inventors have intensively studied the homologous genes of the genes listed in Table 1, have found a substance that regulates its expression, and have completed the invention.

  That is, according to the present invention, the following inventions are provided.

(1) The expression regulator of the gene of Table 1 and / or its homologous gene characterized by containing 2-aminoethanesulfonic acid.
(2) For prevention, diagnosis, improvement or treatment of diseases and symptoms associated with fluctuations in the expression of the genes listed in Table 1 and / or their homologous genes, characterized by containing 2-aminoethanesulfonic acid as an active ingredient Formulation.

  It has been clarified that 2-aminoethanesulfonic acid regulates the expression level of homologous genes of the genes listed in Table 1 under conditions in which the expression of various genes was varied by a running load test system.

One aspect of the present invention is an expression regulator for the genes listed in Table 1 and / or their homologous genes, characterized by containing 2-aminoethanesulfonic acid.
In the present invention, “the genes described in Table 1” means the genes described in Table 1 below.

  The “gene” in the present invention may be either DNA or RNA in a cell, and includes not only genomic DNA but also mRNA, aRNA, cRNA, cDNA and the like. It may be a gene containing an expressed sequence tag (EST).

  Here, the “gene expression regulator” may be either an agent that increases the expression level of a gene or an agent that suppresses gene expression.

  Among the genes listed in Table 1, ACTA2 is a component that constitutes the basic skeleton of cells, and is a molecule that is suggested to be involved in morphology maintenance and tissue repair. CD33 is a sialic acid-dependent cell adhesion molecule present on leukocytes, and is suggested to be involved in tissue damage by leukocytes. CHI3L1 is a molecule that has high homology to chitinolytic enzymes and is implicated in psychiatric and respiratory diseases. DGAT2 is a triacylglycerol synthesis-related enzyme that regulates the production and storage of triglycerides and is a molecule implicated in metabolic diseases. HMBS is an enzyme in the heme biosynthetic pathway and is involved in heme utilization in vivo, and is a molecule that is implicated in erythrocyte function. IFITM1,2 is a molecule whose expression is induced by interferon and is implicated in viral infection and cell proliferation. IL-1β is one of cytokines and is a molecule suggested to be involved in fever during microbial infection and exacerbation of rheumatoid arthritis. MMP8 is a degrading enzyme of type I, II, and III collagens, and is a molecule suggested to be involved in tissue damage and injury. MXD1 has a function of regulating transcription of mRNA, and is a molecule suggested to be involved in diseases accompanied by abnormal cell proliferation. OLFM4 is a molecule that induces cell proliferation and cell death through regulation of the cell cycle and is implicated in diseases with abnormal cell proliferation and cell death. PPP1R3D is a molecule that has the function of regulating the activity of glycogen metabolizing enzymes and is implicated in metabolic diseases and energy production. PRAM1 mediates signal transduction from adhesion molecules and regulates leukocyte function, and is a molecule suggested to be involved in tissue damage by leukocytes. RTP4 has a chaperone function and is a molecule suggested to be involved in intracellular signal transduction. S1000A8 is an intracellular calcium-binding protein that regulates leukocyte function through calcium ion-dependent signal transduction, and is a molecule suggested to be involved in tissue damage by leukocytes. SLFN12 and SLFN12L are cell cycle-related molecules that are induced by interferon and regulate cell growth, and are suggested to be involved in tissue damage caused by infections and leukocytes. STEAP4 is a metal ion-dependent reductase molecule that is implicated in obesity and insulin resistance. TARM1 is a molecule that is expressed on leukocytes and is responsible for signal transduction into cells, suggesting its involvement in tissue damage by leukocytes. TAS2R60 is a molecule that has been reported to associate with sensory receptors in cells and is implicated in taste. TREM1 is a molecule that regulates the production of cytokines and chemokines expressed on leukocytes, and is suggested to be involved in infectious diseases and tissue damage by leukocytes.

  Another aspect of the present invention is the prevention of diseases and symptoms associated with variations in the expression of the genes listed in Table 1 and / or their homologous genes, characterized by containing 2-aminoethanesulfonic acid as an active ingredient. A formulation for diagnosis, amelioration or treatment. Here, “diseases and symptoms associated with changes in the expression of the genes listed in Table 1 and / or their homologous genes” include, for example, type I and type II diabetes, arteriosclerosis, hypertriglyceridemia, Nasu / Hakora Disease, Alzheimer's disease, multiple sclerosis, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, spondyloarthritis, Kawasaki disease, Behcet's disease, systemic lupus erythematosus, irritable colitis, ulcerative colitis, psoriasis vulgaris Disease, scleroderma, acute pancreatitis, lupus nephritis, osteoarthritis, empyema, pneumonia, allergic rhinitis, sinusitis, atopic dermatitis, eczema, bronchial asthma, chronic obstructive pulmonary disease, cystic fibrosis, Pulmonary fibrosis, lung cancer, stomach cancer, liver cancer, breast cancer, colon cancer, colorectal cancer, bladder cancer, prostate cancer, cervical adenocarcinoma, ovarian cancer, glioblastoma, acute myeloid leukemia, acute promyelocytic leukemia Acute phosphorus Blastic leukemia, porphyria, acute intermittent porphyria, thoracic aortic aneurysm, Willis arterial occlusion, ischemic heart disease, coronary artery disease, peptic ulcer disease, fatigue, hepatitis C virus infection, yellow fever virus infection , West Nile virus infection, human immunodeficiency virus infection, dengue virus infection.

  In the present invention, expression variations of homologous genes of the genes listed in Table 1 may be analyzed and / or compared. “Homologous gene” refers to a gene functionally equivalent to a gene selected from the genes listed in Table 1. For some homologous genes, gene information such as base sequences or polypeptide sequences can be easily obtained from genes registered in HGNC, NCBI or the like.

“Functionally equivalent gene” means that the polypeptide encoded by the homologous gene has the same biological function, physiological function or biochemical function as the polypeptide encoded by the gene listed in Table 1. It has shown that.
Usually, functionally equivalent genes have high homology or identity in the base sequence or polypeptide sequence, and are at least 50% or more, preferably 60% or more, more preferably 70% or more, and still more preferably Shows 80% or more, more preferably 90% or more, particularly preferably 95% or more of homology or identity. Functionally equivalent genes can be isolated and identified using a gene amplification method based on the base sequence.

The “gene expression level” in the present invention indicates, for example, the expression level, expression intensity, or expression frequency of a gene product.
The “gene product” may be either a transcription product or a translation product.
“Transcription product” refers to a gene, usually RNA, preferably mRNA, which is produced from a gene through a transcription process.
A “translation product” refers to a protein that is generated by a transcription and translation process from a gene, and may be unmodified or post-translationally modified.

Usually, the expression level of a gene can be analyzed by measuring the expression level of the transcription product or translation product of the gene product, correcting the expression level, and calculating the variation.
As a method for measuring the gene expression level, there are a method using a probe or primer capable of detecting a transcription product, a method using an antibody that recognizes a translation product, or a binding substance.

  In the present invention, “compare with the expression level of a gene” means that the expression level of a specific gene or gene group is measured as described above, and the drug non-administration group and / or the drug administration group and the drug administration group It is shown that the expression level of the gene increases or decreases between the two. The determination of increase or decrease may be qualitative or quantitative.

  The “gene expression regulator” of the present invention can be provided as a reagent for adjusting fluctuations in the product amount of the genes listed in Table 1 and homologous genes thereof, or as a kit containing the same.

  As the “2-aminoethanesulfonic acid” in the present invention, not only a chemically synthesized product, but also 2-aminoethanesulfonic acid extracted from a bloody portion of a fish, shellfish such as oysters and sea bream, and seafood such as squid and octopus is used. be able to.

  The gene expression regulator containing 2-aminoethanesulfonic acid may be a solid (solid agent) or a liquid (liquid agent), and a pH adjuster, a preservative, an antiseptic, and the like can be added as necessary. The reagent or kit may be further combined with a primer, a probe, an antibody or the like for analyzing the expression level of the gene product described in Table 1, if desired.

  The amount of 2-aminoethanesulfonic acid used is, for example, 50 mg to 8000 mg / kg per day, preferably 100 mg to 6000 mg / kg, more preferably 200 mg to 4000 mg / kg when orally administered to mice. A daily dose of 2-aminoethanesulfonic acid can be orally administered once or several times daily.

  Moreover, the “gene expression regulator” of the present invention can be provided as an oral pharmaceutical and / or food characterized by containing 2-aminoethanesulfonic acid. In addition to 2-aminoethanesulfonic acid, these oral compositions include pH adjusters, preservatives, preservatives, excipients, disintegrants, binders, lubricants, as long as the effects of the present invention are not impaired. Agents, antioxidants, coating agents, coloring agents, flavoring agents, cooling agents, suspending agents, antifoaming agents, thickening agents, solubilizing agents, surfactants, fragrances and the like may be added. Furthermore, if necessary, it can be provided as an oral liquid preparation / beverage such as a solid preparation for oral use such as tablets, capsules, powders, granules, dry syrups and the like, or a drink by a conventional method.

  Oral compositions can usually be provided as pharmaceuticals, quasi-drugs, foods for specified health use, etc. that can be used for their indications.

  The human oral dosage of 2-aminoethanesulfonic acid as an oral composition can be appropriately increased or decreased in consideration of age, sex, weight, etc., but is usually 500 mg to 8000 mg per day for adults, and 1000 mg to 6000 mg is preferable, and 3000 mg to 4000 mg is more preferable. A daily dose of 2-aminoethanesulfonic acid can be orally administered once or several times daily.

  Examples of the present invention are specifically shown below, but the present invention is not limited to these examples.

As experimental animals, 7-11 week old BALB / c mice, male (Japan SLC Co., Ltd.) (12 animals) were used. After arrival, the laboratory animals were allowed to freely ingest standard feed (Oriental Yeast Co., Ltd.) and sterilized water throughout the test period and acclimated for at least one week.
The experimental groups were gene expression induction treated mice (4 mice), 2-aminoethanesulfonic acid-administered gene expression induction treated mice (4 mice), and healthy mice (4 mice). Was given.

  Gene expression induction treated mice were produced by gradually increasing forced running load using a mouse / rat treadmill running device (Bioresearch Center Co., Ltd.) (running conditions: inclination angle 10 degrees, running start speed 9 meters / Minute, incremental step 3 meters / 4 minutes, 52 minutes).

  2-Aminoethanesulfonic acid was orally administered at a dose of 300 mg / kg once a day for 2 weeks until the day before the gene expression induction model preparation treatment, and was also orally administered immediately after the completion of the model preparation treatment.

Blood cells were selected as cells. Blood cells were collected from animals by euthanizing mice under deep anesthesia conditions 2 hours after gene expression induction treatment, collecting 1 mL of blood from the abdominal vena cava, adding 1 mL of nuclease-free water, and isogen-LS ( (Nippon Gene) 2 mL was added and the blood was dissolved.

Rough extraction of RNA from blood cells dissolved in Isogen-LS was performed according to the protocol of the reagent. The crudely extracted RNA was subjected to column purification using RNeasy Mini Kit (Qiagen).
After quantification and quality check of TOTAL RNA after column purification, after removal of globin mRNA that is contained in blood in large amounts from necessary and sufficient amount of RNA and hinders expression analysis Again, quantitative and quality checks were performed. For removing globin mRNA, GLOBINclear-Mouse / Rat (Ambion) was used.
Furthermore, the total RNA concentration was measured using NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific). Thereafter, electrophoresis was performed using Agilent RNA 6000 Nano Kit (Agilent Technologies) and Agilent 2100 Bioanalyzer (Agilent Technologies) to measure the RIN value of total RNA.

A reverse transcription reaction solution was prepared, and RNA reverse transcription reaction was performed using GeneAmp PCR System 9700 (Life Technologies).
Next, according to the BioMark 96.96 dynamic array device and the protocol attached to the reagent, Sample mix was applied to Sample mix, Assay mix was applied to Assay inlets, and real-time PCR reaction was performed using BioMark. The maximum number of PCR reactions was 35 cycles. The measurement was repeated three times.

Comparison of the expression level of each gene is based on the number of cycles (threshold cycle value: Ct value) resulting in a constant amount of amplification product obtained by real-time PCR, using the β-actin gene (Actb) as an internal standard gene. The value was calculated from the relative value when the value of the sample was 1.00, and the gene expression level (average value) in blood cells of healthy mice was calculated as 100%.
As PCR primers for each gene and β-actin gene, wet validation primers commercially available from BioMark were used.

Expression levels of the genes shown in Table 1 in blood cells derived from gene expression-fluctuated mice were significantly increased compared to healthy mice.
The expression levels of the mouse homologous genes of the genes described in Table 1 in the gene expression induction treated mice administered with 2-aminoethanesulfonic acid were clearly compared with those in the gene expression induction treated mice not administered with 2-aminoethanesulfonic acid. It was decreasing. Specific fluctuation amounts are shown in Table 2.

健常非運動負荷マウスの発現量を100(%)とし、運動負荷による増加量を記載した。その値をもとに、２−アミノエタンスルホン酸投与による減少率を算出した。

The expression level of healthy non-exercise mice was defined as 100 (%), and the increase due to exercise load was described. Based on the value, the reduction rate by administration of 2-aminoethanesulfonic acid was calculated.

  From the above, 2-aminoethanesulfonic acid attenuated the increase in the expression of homologous genes of the genes described in Table 1 in the blood cells derived from the gene expression variation treated mice, and regulated the expression variation.

  According to the present invention, it is possible to provide reagents, pharmaceuticals, and foods that regulate fluctuations in the expression level of the genes listed in Table 1 and / or their homologous genes.

Claims (2)

The expression regulator of the gene of Table 1 and / or its homologous gene characterized by containing 2-aminoethanesulfonic acid. A preparation for preventing, diagnosing, improving or treating diseases and symptoms associated with fluctuations in the expression of the genes shown in Table 1 and / or homologous genes thereof, comprising 2-aminoethanesulfonic acid as an active ingredient.