Ammonia-reducing components remove senescent cells

Abstract

To develop a method that can conveniently remove senescent cells within an organism with few side effects.SOLUTION: The present inventors focused on the mechanism by which the ammonia metabolically produced in excess maintains the survival of the senescent cell, and demonstrated that senolysis is made possible by enhancing ammonia excretion. More specifically, it was demonstrated in animal experiments with mice that the expression level of p21, a senescent cell marker, is significantly lowered when an ammonia-excretion component is administered to aged mice. It was shown for the first time that a senolytic effect is exhibited by the intake of a component that removes ammonia.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to providing a new means for removing senescent cells from the living body and preventing aging. More specifically, it relates to providing a new means for removing senescent cells from the living body and preventing aging by a substance having an ammonia excretion promoting effect in the living body.

Background Art

[0002] In the human body, "senescent cells" which are cells that have become unable to proliferate with aging accumulate in the body, and are known to cause diseases such as arteriosclerosis and fatty liver and aging phenomena. Also, in recent research, when mice capable of removing senescent cells were created, it became clear that the onset of age-related pathological conditions was delayed (Non-Patent Document 1, Non-Patent Document 2).

[0003] It has been reported that by removing senescent cells (senolysis), it is possible to prevent or delay physical dysfunction and extend the healthy life span (Non-Patent Document 1).

[0004] However, many of the candidate drugs for removing senescent cells reported so far are those used as anticancer agents, and there have been concerns that the side effects are too strong for use in removing senescent cells.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] The objective of this invention is to develop a method that can remove senescent cells in the body or prevent aging in a simpler manner with fewer side effects. [Means for solving the problem]

[0007] The inventors focused on the mechanism by which excess ammonia produced by metabolism maintains the survival of senescent cells, and hypothesized that promoting the excretion of ammonia in the body might remove senescent cells or prevent aging. Therefore, they decided to investigate, using aged mice, whether drugs and food components known to promote ammonia excretion have the effect of removing senescent cells or preventing aging.

[0008] The inventors focused on the mechanism by which excess ammonia produced by metabolism maintains the survival of senescent cells and demonstrated that senescent cells can be removed or aging prevented by promoting ammonia excretion. More specifically, in animal experiments using mice, they revealed that administering an ammonia-excreting component to aged mice significantly reduced the expression level of p21, a marker for senescent cells. This was the first time that the intake of an ammonia-removing component exerted a senescent cell-removing effect.

[0009] More specifically, this application provides the following embodiments to solve the aforementioned problems: [1] A senescent cell remover containing a substance that promotes ammonia excretion in the body; [2]: The senescent cell scavenger according to [1], wherein the substance having an ammonia excretion promoting effect in the body is one or more substances selected from the group consisting of ornithine, citrulline, taurine, carnitine, arginine, branched-chain amino acids (BCAAs), acetylglutamic acid, sodium benzoate, lactulose, lactitol, sodium phenylbutyrate, sodium phenylacetate, rifaximin, and cargrumic acid; [3]: The senescent cell scavenger according to [1], wherein the substance that promotes ammonia excretion in the body is lactulose, ornithine, sodium benzoate, or any combination thereof; [4]: Anti-aging agents containing substances that promote ammonia excretion in the body. [5]: The anti-aging agent according to [4], wherein the substance that promotes ammonia excretion in the body is one or more substances selected from the group consisting of ornithine, citrulline, taurine, carnitine, arginine, branched-chain amino acids (BCAAs), acetylglutamic acid, sodium benzoate, lactulose, lactitol, sodium phenylbutyrate, sodium phenylacetate, rifaximin, and cargrumic acid. [6]: The anti-aging agent described in [4], wherein the substance that promotes ammonia excretion in the body is lactulose, ornithine, sodium benzoate, or any combination thereof. [7]: Compositions containing substances that promote ammonia excretion in the body and that have senescent cell removal or anti-aging effects; [8]: The composition described in [7], which is for medicinal use; [9]: The composition according to [7] or [8], wherein the substance having an ammonia excretion-promoting effect in the body is one or more substances selected from the group consisting of ornithine, citrulline, taurine, carnitine, arginine, branched-chain amino acids (BCAAs), acetylglutamic acid, sodium benzoate, lactulose, lactitol, sodium phenylbutyrate, sodium phenylacetate, rifaximin, and cargrumic acid;

[10] : The composition according to [7] or [8], wherein the substance that promotes ammonia excretion in the body is lactulose, ornithine, sodium benzoate, or any combination thereof. [Effects of the Invention]

[0010] The present invention can provide a senescent cell scavenger or anti-aging agent, or a composition having a senescent cell scavenger or anti-aging effect, which contains a substance that promotes ammonia excretion in the body. This senescent cell scavenger or anti-aging agent, or composition having a senescent cell scavenger or anti-aging effect, can provide a method for removing senescent cells in the body and preventing aging in a simpler way, by simply taking it orally, without the side effects of anticancer drugs. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows the results of measuring the mRNA expression level of p21, a marker of senescent cells, in the kidney. [Figure 2] Figure 2 shows the results of measuring the mRNA expression levels of p21 and p16, which are markers of senescent cells, in the liver. [Figure 3] Figure 3 shows the results of measuring the mRNA expression level of p16, a marker for senescent cells, in the hippocampus. [Figure 4] Figure 4 shows the results of measuring the mRNA expression level of p16, a marker for senescent cells, in skeletal muscle. [Modes for carrying out the invention]

[0012] The inventors of this invention focused on the mechanism by which excess ammonia produced by metabolism maintains the survival of senescent cells, and demonstrated that senescent cells can be removed by promoting ammonia excretion.

[0013] As a result, in a first aspect of this invention, the present invention can provide a senescent cell removal agent containing a substance that promotes ammonia excretion in the body.

[0014] Generally, senescent cells refer to cells that have irreversibly stopped cell division due to damage to chromosomal DNA caused by various stresses such as telomere abnormalities, oxidative stress, DNA damage, activation of oncogenes, mitochondrial dysfunction, viral infection, and inflammation. In senescent cells, it is known that the p16 gene and p21 gene are highly expressed, and these genes are regarded as representative markers of senescent cells. Functionally, the accumulation of these senescent cells in the body leads to the high expression and extracellular secretion of various inflammatory proteins such as cytokines and chemokines, resulting in functional decline of various tissues, chronic inflammation, carcinogenesis, etc.

[0015] Based on such characteristics, it has been proposed that removing senescent cells with senolytic drugs can improve aging and geriatric diseases (Non-Patent Document 1).

[0016] Common characteristics of senescent cells include that along with the accumulation of misfolded proteins, damage occurs to the intracellular lysosomal membrane, and the contents with a pH of around 5 in the lumen of lysosomes leak into the cell, resulting in a decrease in intracellular pH. In such cells, the production of glutaminase 1 (GLS1), which is a glutamine metabolic enzyme, is activated, leading to excessive production of glutamic acid and ammonia from glutamine. It is considered that the ammonia produced in excess regulates the constancy of intracellular pH to maintain survival.

[0017] Therefore, it was considered that promoting the removal of senescent cells by removing ammonia from the cell and acidifying the intracellular pH of senescent cells so that they cannot survive.

[0018] There are several pathways for ammonia excretion in cells. Specifically, · The urea cycle (ornithine cycle) that finally generates urea from the reaction of ornithine or citrulline with ammonia to detoxify and excrete ammonia • Acid amide formation: The reaction of glutamic acid (Glu) or aspartic acid (Asp) with ammonia produces glutamine (Gln) or asparagine (Asn). • Reaction of α-ketoglutaric acid with ammonia to produce glutamic acid (Glu) • Creatine production • A pathway that lowers blood ammonia levels by activating the urea cycle. • A pathway that lowers ammonia levels in the blood by acting on intestinal bacteria to reduce ammonia production. • A pathway in which it combines with ammonia and is excreted from the body as hippuric acid. Several ammonia emission pathways are known.

[0019] In the present invention, a substance that can be used as an active ingredient and has an ammonia excretion promoting effect in the body refers to a substance that can act on one or more of the above-mentioned ammonia excretion pathways to promote the excretion of ammonia. For example, substances that react with ammonia, such as ornithine or citrulline, which are components of the ornithine cycle, glutamic acid or aspartic acid in acid amide production, and α-ketoglutaric acid in reactions with keto acids, can be used for this purpose, but are not limited to these.

[0020] More specifically, examples of substances that can be used include, but are not limited to, ornithine, citrulline, taurine, carnitine, arginine, branched-chain amino acids (BCAAs), acetylglutamic acid, sodium benzoate, lactulose, lactitol, sodium phenylbutyrate, sodium phenylacetate, rifaximin, and cargrumic acid. For example, any substance currently being used in clinical research to treat hyperammonemia may also be used in the future.

[0021] In the present invention, in a preferred embodiment, lactulose, ornithine, sodium benzoate, or any combination thereof can be used as the substance having the above-mentioned ammonia excretion promoting effect in the body.

[0022] In addition to the above-mentioned active ingredients, the senescent cell removal agent of the present invention may also contain various additives commonly used in food or pharmaceuticals.

[0023] In the present invention, the removal of senescent cells can include inducing cell death in senescent cells and removing them from the body, or altering the cellular properties of senescent cells to reduce their characteristic features. Confirmation of whether senescent cells have been removed can be performed by checking for a decrease in the expression of p16 and p21 genes, which are characteristic of senescent cells, a decrease in the activity of senescence-related acid β-galactosidase (SAβ-Gal), and cell staining (SAβ-Gal staining, HE staining, PAS staining, Sirius Red staining, Masson's trichrome staining) in a sample taken from a living organism.

[0024] The amount of active ingredient ingested in this invention varies depending on the substance that can be used as an active ingredient, and can be included in amounts commonly used for each substance. The intake schedule for each active ingredient can be anything; for example, it may be taken once a day, three times a day, or at a reduced frequency, such as once a week or once a month.

[0025] As a derivative of the first aspect of the present invention, an anti-aging agent can be provided that contains a substance that promotes ammonia excretion in the body. In the present invention, as described above, aging can be prevented by removing senescent cells with the active ingredient, which is a substance that promotes ammonia excretion in the body.

[0026] In a second aspect of the present invention, the present invention can provide a composition having a senescent cell removal effect or an anti-aging effect, which contains a substance that promotes ammonia excretion in the body.

[0027] As mentioned above, the substances that can be used as active ingredients in the present invention and have an ammonia excretion promoting effect in the body include substances that exist in the body, substances contained in food, and substances that require approval as pharmaceuticals. Therefore, when referring to a composition in this embodiment, it may be a food composition (a composition for general food products, and a composition for foods with biological regulatory functions (for example, Foods for Specified Health Uses, Foods with Nutrient Function Claims, or Foods with Function Claims)) or a pharmaceutical composition.

[0028] In the composition of the present invention, in addition to the above-mentioned active ingredient (a substance that promotes ammonia excretion in the body), other ingredients and various additives commonly used in food or pharmaceuticals may be included, and any of these other ingredients and additives are acceptable.

[0029] When using a pharmaceutical composition containing a substance that promotes ammonia excretion in the body and has a senescent cell removal effect or senescence prevention effect, it is common to administer it orally. However, the method of administration can be changed depending on the site where the senescent cells to be removed are located, and administration can also be done using routes other than oral administration.

[0030] For example, when the objective is to remove senescent cells or prevent aging in tissues accessible via the bloodstream, the active ingredient of the present invention can be administered using routes of administration such as oral administration, intravenous injection, subcutaneous injection, direct intratissue administration, or intraperitoneal administration.

[0031] For example, when the goal is to remove senescent cells in the brain, which are difficult to access directly via the bloodstream, or to prevent aging, the drug can be administered using routes such as epidural administration, intraventricular administration, or nasal administration.

[0032] The present invention will be specifically illustrated below with reference to examples. The examples shown below do not limit the present invention in any way. [Examples]

[0033] Example 1: Investigation of the effect of removing senescent cells In this example, various substances having ammonia excretion-promoting effects were administered to animals to confirm whether each substance had the effect of removing senescent cells.

[0034] (1) Sample preparation For the animals used, seven 7-week-old male C57BL / 6J mice were used as young mice, and 28 78-week-old male C57BL / 6J-Aged mice were used as aged mice in the experiment.

[0035] Each animal was raised in an SPF (Specific Pathogen Free) environment with a temperature of 20-26°C, humidity of 30-70%, and lighting for 12 hours a day (lights on at 7:00 AM and off at 7:00 PM). Temperature and humidity were recorded daily and stored as raw data.

[0036] The animals were kept individually in polycarbonate mouse cages (170 W x 240 D x 120 H mm, manufactured by Nippon Crea Co., Ltd.). Paper chips, specifically Pepperclean (manufactured by Nippon SLC Co., Ltd.), were used as bedding, sterilized in an autoclave (temperature 121°C, sterilization 40 minutes, drying 30 minutes).

[0037] The mice and rats were given free access to solid feed MF (manufactured by Oriental Yeast Co., Ltd.), which had been autoclaved (temperature 121°C, sterilization 15 minutes, drying 5 minutes).

[0038] Water was supplied using 100 mL polycarbonate water bottles, with 0.025% sodium hypochlorite (Tsurukuron, manufactured by Tsurumi Soda Co., Ltd.) added to the locally pumped water, which was then made available for free intake.

[0039] Young mice were designated as Group 1, and older mice were divided into four groups of seven (Groups 2-5), with the experimental conditions assigned to each group as shown in the table below.

[0040] [Table 1]

[0041] As explained below, groups 3 through 5 were given water to which the test substance had been added. The test substance administered to groups 3 through 5 was prepared as follows: component A for group 3, component B for group 4, and component C for group 5. The test substance was prepared twice a week. • Component A: L-ornithine hydrochloride (99%, Sigma-Aldrich) was added to water to a concentration of 1% to prepare the solution. • Component B: L-ornithine hydrochloride (99%, Sigma-Aldrich) was added to water to a concentration of 1%, and sodium benzoate (>99%, Sigma-Aldrich) was added to water to a concentration of 0.0006%. • Component C: Lactulose syrup (60%, Kowa Pharmaceutical) was added to water to a concentration of 7.2% to prepare the solution.

[0042] Drinking water prepared by adding the test substance using these methods was given to each group free of charge and administered orally for one month. One month after the start of the experiment, all animals were euthanized and dissected, and the liver, kidneys, hippocampus, and skeletal muscle were collected from each animal to measure markers of senescent cells.

[0043] (2) Measurement of markers in senescent cells (2-1) RNA extraction Tissue samples taken from mice during dissection were immersed overnight in RNA later (Ambion) and then stored at -80°C. Total RNA was extracted from liver tissue using the TRIzol-chloroform method.

[0044] 50-100 mg of tissue isolated from RNA later was placed in 1 ml of TRIzol Reagent (Invitrogen Life Technologies) and ground. After standing at room temperature for 5 minutes, 0.2 ml of chloroform was added and mixed, and after standing at room temperature for 2-3 minutes, the mixture was centrifuged at 4°C and 12,000 × g for 15 minutes. 250 μl of the upper layer was collected and 0.5 ml of isopropanol was added. After standing at room temperature for 10 minutes, the mixture was centrifuged again at 4°C and 12,000 × g for 10 minutes.

[0045] The RNA was purified with 75% ethanol, evaporated to dryness under reduced pressure, and then dissolved in 50 μl of RNA-free water. RNA purity was confirmed by measuring the absorbance ratio (A260 / 280 nm) at 260 nm and 280 nm using NanoDrop ND-1000 (NanoDrop Technologies) and confirming that the value fell within the range of 1.9 to 2.1.

[0046] (2-2) Real-time RT-PCR Using total RNA extracted from tissue, mRNA expression analysis was performed on p16 and p21, which are commonly known aging markers in this field, by quantitative real-time PCR.

[0047] cDNA was synthesized from total RNA using the PrimeScript® RT reagent Kit (Perfect Real Time) according to the manufacturer's protocol. The cDNA was amplified using the Thermal Cycler Dice Real Time System TP800 (Takara Bio) and then processed using SYBR® Premix Ex Taq. TM Fluorescence was detected using the Perfect Real Time and real-time PCR detection system (Takara Bio).

[0048] The PCR reaction was performed as follows: one cycle at 95°C for 10 seconds, followed by 40 cycles of [95°C for 5 seconds, 60°C for 30 seconds], and then one cycle of [95°C for 15 seconds, 60°C for 30 seconds, 95°C for 15 seconds].

[0049] The mRNA expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard for correction, and the relative value of the target mRNA was calculated. Each primer was designed using the web application PRIMER3, and its synthesis was commissioned to Invitrogen. The primer sequences for gene amplification of the target genes, the aging markers p16 and p21, are shown below.

[0050] [ka]

[0051] (2-3) Effect of removing senescent cells In the liver, kidney, hippocampus, and skeletal muscle tissue samples collected in (1) above, the expression of the aging markers p16 and p21 was confirmed by PCR amplification, as described in (2-1) and (2-2). In the figure shown below, "*" indicates p<0.05, "**" indicates p<0.01, and "***" indicates p<0.005.

[0052] (2-3-1) Expression levels in the kidneys In the kidneys, when the mRNA expression level of p21, a marker of senescent cells, was measured, it was significantly reduced in the group that ingested lactulose (labeled "Aged+L" in the figure) compared to aged mice that received no treatment (labeled "Aged" in the figure) (Figure 1).

[0053] (2-3-2) Expression levels in the liver In the liver, when the mRNA expression level of p21, a marker of senescent cells, was measured, p21 mRNA expression levels were significantly reduced in all experimental groups (ornithine intake group, ornithine + sodium benzoate group, lactulose intake group) compared to aged mice that received no treatment (labeled "Aged" in the figure) (Figure 2, upper panel). In particular, the reduction was significant in the ornithine + sodium benzoate intake group (labeled "Aged+OB" in the figure), and showed a decreasing trend in the ornithine intake group (labeled "Aged+O" in the figure) and the lactulose intake group (labeled "Aged+L" in the figure) (p<0.1).

[0054] In the liver, we also measured the mRNA expression level of p16, a marker of senescent cells. Compared to aged mice that received no treatment (labeled "Aged" in the figure), the ornithine-supplemented group (labeled "Aged+O" in the figure) showed a decreasing trend in p16 mRNA expression, while the lactulose-supplemented group (labeled "Aged+L" in the figure) showed a significant decrease in p16 mRNA expression (Figure 2, lower panel).

[0055] (2-3-3) Expression levels in the hippocampus In the hippocampus, when the mRNA expression level of p16, a marker of senescent cells, was measured, although not statistically significant, p16 mRNA expression levels were reduced in all experimental groups (ornithine intake group (labeled "Aged+O" in the figure), ornithine + sodium benzoate intake group (labeled "Aged+OB" in the figure), and lactulose intake group (labeled "Aged+L" in the figure)) compared to aged mice that received no treatment (labeled "Aged" in the figure) (Figure 3).

[0056] (2-3-4) Expression levels in skeletal muscle In skeletal muscle, when the mRNA expression level of p16, a marker of senescent cells, was measured, although not statistically significant, p16 mRNA expression levels were reduced in all experimental groups (ornithine intake group (labeled "Aged+O" in the figure), ornithine + sodium benzoate intake group (labeled "Aged+OB" in the figure), and lactulose intake group (labeled "Aged+L" in the figure)) compared to aged mice that received no treatment (labeled "Aged" in the figure) (Figure 4). [Industrial applicability]

[0057] The present invention can provide a senescent cell scavenger or a composition having a senescent cell scavenger effect, which contains a substance that promotes ammonia excretion in the body. This senescent cell scavenger or composition has a senescent cell scavenger effect provides a method for removing senescent cells in the body in a simpler way, by simply taking it orally, without the side effects of anticancer drugs.

Claims

1. A senescent cell remover comprising ornithine, and one or more substances selected from the group consisting of sodium benzoate, lactulose, and lactitol, which are used in combination with ornithine, and which have an ammonia excretion promoting effect in the body.

2. A composition for removing senescent cells, comprising ornithine, and one or more substances selected from the group consisting of sodium benzoate, lactulose, and lactitol used in combination with ornithine, which have an ammonia excretion promoting effect in the body.

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