Senolysis by ammonia-lowering component

Abstract

To develop means for removing senescent cells present in a living body with a simpler method with fewer side effects.SOLUTION: The inventors of the present invention focused on a mechanism of maintaining the survival of senescent cells by ammonia which is generated in excess through metabolism and demonstrated that the promotion of the ammonia excretion enables the removal of senescent cells. The present invention provides an agent for removing senescent-cells, comprising one or more substances having an action for promoting ammonia excretion in the body, selected from the group consisting of ornithine, citrulline, taurine, carnitine, arginine, branched-chain amino acids (BCAAs), acetyl glutamic acid, sodium benzoate, sodium phenylbutyrate, sodium phenylacetate, carglumic acid, lactulose, lactitol, and rifaximin.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to providing a new means for removing senescent cells from a living body and preventing aging. More specifically, the present invention relates to providing a new means for removing senescent cells from a living body and preventing aging by using a substance that has an action of promoting ammonia excretion in a living body. [Background technology]

[0002] It is known that "senescent cells," which are cells that can no longer proliferate with age, accumulate in the human body and cause diseases and aging phenomena such as arteriosclerosis and fatty liver. Furthermore, recent research has revealed that when mice in which senescent cells can be removed were created, the onset of age-related pathologies was delayed (Non-Patent Document 1, Non-Patent Document 2).

[0003] It has been reported that removing senescent cells (senolysis) can prevent or delay bodily dysfunction and extend healthy lifespan (Non-Patent Document 1).

[0004] However, many of the candidate drugs for removing senescent cells that have been reported so far are used as anticancer drugs, and there have been concerns that their side effects are too strong for their use in removing senescent cells. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Johmura Y., SCIENCE, Vol 371, Issue 6526, pp. 265-270, 2021 [Non-Patent Document 2] Suda M., et al., Nature Aging, vol 1, pages 1117-1126, 2021 Summary of the Invention [Problem to be solved by the invention]

[0006] An objective of the present invention is to develop a method for removing senescent cells or preventing aging in a living body more simply and with fewer side effects. [Means for solving the problem]

[0007] The inventors focused on the mechanism by which ammonia produced in excess through metabolism maintains the survival of senescent cells, and hypothesized that promoting the excretion of ammonia in the body might enable the elimination of senescent cells or the prevention of aging. They therefore decided to use aged mice to verify whether drugs and food ingredients known to promote the excretion of ammonia also have the effect of eliminating senescent cells or preventing aging.

[0008] The inventors have focused on the mechanism by which excess ammonia produced by metabolism maintains the survival of senescent cells, and demonstrated that promoting ammonia excretion can eliminate senescent cells or prevent aging. More specifically, in animal experiments using mice, they have demonstrated that the expression level of p21, a marker of senescent cells, is significantly reduced when a component that excretes ammonia is administered to aged mice. This is the first time that they have demonstrated that the intake of a component that removes ammonia has the effect of eliminating senescent cells.

[0009] More specifically, in order to solve the above-mentioned problems, the present application provides the following aspects: [1]: A senescent cell remover containing a substance that promotes ammonia excretion in the body; [2]: the senescent cell removing agent according to [1], wherein the substance having an effect of promoting 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 carglumic acid; [3]: the senescent cell removing agent according to [1], wherein the substance having an ammonia excretion promoting effect in the body is lactulose, ornithine, sodium benzoate, or any combination thereof; [4]: An anti-aging agent containing a substance that promotes ammonia excretion in the body. [5]: The anti-aging agent according to [4], 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 carglumic acid. [6]: The anti-aging agent according to [4], wherein the substance having the effect of promoting ammonia excretion in the body is lactulose, ornithine, sodium benzoate, or any combination thereof. [7]: a composition having an effect of removing senescent cells or preventing aging, comprising a substance having an effect of promoting ammonia excretion in the body; [8]: The composition according to [7], which is for pharmaceutical 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 carglumic acid;

[10] : The composition described in [7] or [8], wherein the substance having an ammonia excretion promoting effect in the body is lactulose, ornithine, sodium benzoate, or any combination thereof. Effect of the Invention

[0010] The present invention can provide a senescent cell removing agent or anti-aging agent, or a composition having a senescent cell removing or anti-aging effect, which contains a substance that has an effect of promoting ammonia excretion in the body. This senescent cell removing agent or anti-aging agent, or a composition having a senescent cell removing or anti-aging effect, can provide a method for removing senescent cells in the body and preventing aging in a simpler manner of merely taking it orally, without side effects like anti-cancer drugs. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows the results of measuring the expression level of p21 mRNA, a marker for senescent cells, in the kidney. [Diagram 2] FIG. 2 shows the results of measuring the levels of mRNA expression of p21 and p16, which are markers for senescent cells, in the liver. [Diagram 3] FIG. 3 shows the results of measuring the expression level of p16 mRNA, a marker for senescent cells, in the hippocampus. [Figure 4] FIG. 4 shows the results of measuring the expression level of mRNA of p16, a marker for senescent cells, in skeletal muscle. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The inventors of the present invention focused on the mechanism by which ammonia produced in excess 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, the present invention can provide an agent for removing senescent cells, which comprises a substance that has the effect of promoting ammonia excretion in the body.

[0014] In general, senescent cells are 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 cancer genes, mitochondrial dysfunction, viral infection, and inflammation. It is known that p16 and p21 genes are highly expressed in senescent cells, and these genes are considered to be representative markers of senescent cells. In addition, functionally, it is known that 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, which causes functional decline in various tissues, chronic inflammation, carcinogenesis, etc.

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

[0016] A common characteristic of aging cells is that the accumulation of defective proteins causes damage to the intracellular lysosomal membrane, causing the contents of the lysosomal lumen, which have a pH of around 5, to leak into the cell, lowering the intracellular pH. In such cells, production of the glutamine metabolic enzyme glutaminase 1 (GLS1) is activated, resulting in the excessive production of glutamic acid and ammonia from glutamine. It is thought that this excess ammonia regulates the homeostasis of the intracellular pH and maintains survival.

[0017] Therefore, the researchers hypothesized that removing ammonia from within cells would acidify the intracellular pH of senescent cells, thereby making them unable to survive, thereby promoting the removal of senescent cells.

[0018] There are several pathways for excreting ammonia within cells. Specifically, The urea cycle (ornithine cycle) in which ornithine or citrulline reacts with ammonia to produce urea, which is then detoxified and excreted. Acid amide formation from the reaction of glutamic acid (Glu) or aspartic acid (Asp) with ammonia to produce glutamine (Gln) or asparagine (Asn) Reaction of α-ketoglutaric acid with ammonia to produce glutamic acid (Glu) Creatine production · Activation of the urea cycle to lower blood ammonia levels A pathway that reduces blood ammonia levels by acting on intestinal bacteria to reduce ammonia production Binding with ammonia and excreting it from the body as hippuric acid The following ammonia excretion pathways are known:

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

[0020] More specifically, for example, ornithine, citrulline, taurine, carnitine, arginine, branched chain amino acids (BCAA), acetylglutamic acid, sodium benzoate, lactulose, lactitol, sodium phenylbutyrate, sodium phenylacetate, rifaximin, carglumic acid, etc. can be used, but are not limited to these. For example, any substance currently being used to treat hyperammonemia that is currently undergoing clinical research can also be used in the future.

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

[0022] The senescent cell removing agent of the present invention may contain, in addition to the above-mentioned active ingredients, various additives that are commonly used in foods or medicines.

[0023] In the present invention, the removal of senescent cells can include removing senescent cells from the body by inducing cell death in the senescent cells and reducing the characteristics of senescent cells by changing the cellular properties of the senescent cells. Whether or not senescent cells have been removed can be confirmed by confirming a decrease in the expression of p16 gene and p21 gene, which are characteristics of senescent cells, a decrease in the activity of senescence-associated 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 collected from the body.

[0024] The amount of the active ingredient in the present invention varies depending on each substance that can be used as an active ingredient, and each substance can be included in the amount generally used for each substance.The intake schedule for each active ingredient may be any schedule, for example, once a day, three times a day, or less frequently, such as once a week or once a month.

[0025] As a derivative of the first aspect, the present invention can provide an anti-aging agent comprising a substance having an action of promoting ammonia excretion in a living body. As described above, in the present invention, aging can be prevented by removing senescent cells with the substance having an action of promoting ammonia excretion in a living body, which is an active ingredient.

[0026] In a second aspect, the present invention can provide a composition having an effect of removing senescent cells or preventing aging, which comprises a substance having an effect of promoting ammonia excretion in the body.

[0027] As described above, substances having an action of promoting ammonia excretion in the living body that can be used as an active ingredient in the present invention include substances present in the living body, substances contained in food, substances that need to be approved as medicines, etc. Therefore, when referring to a composition in this embodiment, it may be a food composition (a composition for general food and a composition for food having a biological regulation function (e.g., a food for specified health uses, a food with nutritional function claims, or a food with functional claims)) or a pharmaceutical composition.

[0028] The composition of the present invention may contain, in addition to the above-mentioned active ingredient (a substance having an effect of promoting ammonia excretion in the body), other ingredients and various additives that are generally used in foods or medicines, and these other ingredients and various additives are acceptable regardless of their types.

[0029] When a pharmaceutical composition containing a substance that promotes ammonia excretion in the body and has a senescent cell removing or anti-aging effect is used, it is generally administered orally. However, the method of administration can be changed depending on the site where the senescent cells to be removed are present, and administration routes other than oral administration can also be used.

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

[0031] For example, when the objective is to remove senescent cells or prevent senescence in the brain, which is difficult to access directly via blood, the compound can be administered using administration routes such as epidural administration, intraventricular administration, and intranasal administration.

[0032] The present invention will be described in more detail below with reference to examples, but the following examples are not intended to limit the present invention in any way. EXAMPLES

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

[0034] (1) Sample preparation Regarding the animals, the experiment was performed using seven 7-week-old male C57BL / 6J mice as young mice and 28 78-week-old male C57BL / 6J-Aged mice as old mice.

[0035] Each animal was raised in an SPF environment with a temperature of 20-26°C, humidity of 30-70%, and lighting time of 12 hours / day (lights on at 7:00, lights off at 19:00). Temperature and humidity were recorded daily and stored as raw data.

[0036] The animals were individually housed in polycarbonate mouse cages (170 W × 240 D × 120 H mm, manufactured by CLEA Japan Co., Ltd.) for breeding. Paper chips and Peperclean (manufactured by Japan SLC Co., Ltd.) sterilized by autoclave (temperature 121°C, sterilization for 40 minutes, drying for 30 minutes) were used for bedding.

[0037] The mice were given autoclave-sterilized (temperature 121°C, sterilization for 15 minutes, drying for 5 minutes) solid mouse / rat feed MF (manufactured by Oriental Yeast Co., Ltd.) ad libitum.

[0038] Water was provided in 100 mL polycarbonate water bottles, and 0.025% sodium hypochlorite (Tsurukuron, Tsurumi Soda Co., Ltd.) was added to the home-pumped water, which was available ad libitum.

[0039] The young mice were grouped into group 1, and the old mice were divided into four groups of seven mice each (groups 2 to 5), and the experimental conditions were assigned as shown in the table below.

[0040] [Table 1]

[0041] As explained below, groups 3 to 5 were given water containing test substances. The test substances to be administered to groups 3 to 5 were prepared as follows: component A for group 3, component B for group 4, and component C for group 5. The test substances were prepared twice a week: Component A: L-ornithine hydrochloride (99%, Sigma-Aldrich) was added to water to make a 1% solution. Component B: Prepared by adding L-ornithine hydrochloride (99%, Sigma-Aldrich) to water to make a concentration of 1% and sodium benzoate (>99%, Sigma-Aldrich) to water to make a concentration of 0.0006%. Component C: Lactulose syrup (60%, Kowa Pharmaceuticals) was added to water to make a 7.2% solution.

[0042] The test substances were added to the drinking water prepared by these methods and administered orally to each group ad libitum for one month. One month after the start of the experiment, all animals were sacrificed and dissected, and the liver, kidney, hippocampus, and skeletal muscle were collected from each animal to measure markers of senescent cells.

[0043] (2) Measurement of senescent cell markers (2-1) RNA extraction Tissues collected from mice at the time of autopsy were soaked in RNA later (Ambion) overnight and then stored at -80°C. Total RNA was extracted from liver tissue using the TRIzol-chloroform method.

[0044] 50-100 mg of tissue extracted from RNA later was placed in 1 ml of TRIzol Reagent (Invitrogen Life Technologies) and crushed. After standing at room temperature for 5 minutes, 0.2 ml of chloroform was added and stirred, and after standing at room temperature for 2-3 minutes, it 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, it 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 dissolved in 50 μl of RNA-free water. The absorbance ratio at 260 nm and 280 nm (A260 / 280 nm) was measured using a NanoDrop ND-1000 (NanoDrop Technologies) and the RNA purity was confirmed by confirming that the value was within the range of 1.9 to 2.1.

[0046] (2-2) Real-time RT-PCR Using total RNA extracted from the tissues, mRNA expression analysis was performed by quantitative real-time PCR for p16 and p21, which are generally known in the art as senescence markers.

[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 purified using SYBR® Premix Ex Taq TM Fluorescence was detected using a Perfect Real Time (Perfect Real Time) and real-time PCR detection system (Takara Bio).

[0048] The PCR reaction consisted of one cycle of 95°C for 10 sec, followed by 40 cycles of [95°C for 5 sec, 60°C for 30 sec], and then one cycle of [95°C for 15 sec, 60°C for 30 sec, 95°C for 15 sec].

[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 synthesis was requested to Invitrogen. The primer sequences for amplifying the target genes, p16 and p21, which are aging markers, are shown below.

[0050] [ka]

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

[0052] (2-3-1) Expression in the kidney When the mRNA expression level of p21, a marker of senescent cells, was measured in the kidneys, it was significantly reduced in the group that had consumed lactulose (labeled "Aged+L" in the figure) compared to the old mice that had not been administered anything (labeled "Aged" in the figure) (Figure 1).

[0053] (2-3-2) Expression in the liver When the expression level of p21 mRNA, a marker of senescent cells, was measured in the liver, it was found to be significantly decreased in all experimental groups (the ornithine-fed group, the ornithine + sodium benzoate group, and the lactulose-fed group) compared with the untreated aged mice (labeled "Aged" in the figure) (Figure 2, upper panel). In particular, it was significantly decreased in the ornithine + sodium benzoate-fed group (labeled "Aged + OB" in the figure), and tended to decrease in the ornithine-fed group (labeled "Aged + O" in the figure) and the lactulose-fed 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 for senescent cells.Compared to old mice that had not been administered anything (labeled "Aged" in the figure), the mRNA expression level of p16 tended to decrease in the ornithine-fed group (labeled "Aged+O" in the figure), and the mRNA expression level of p16 was significantly decreased in the lactulose-fed group (labeled "Aged+L" in the figure) (lower panel of Figure 2).

[0055] (2-3-3) Expression in the hippocampus When the mRNA expression level of p16, a marker of aging cells, was measured in the hippocampus, it was found that, although not significantly, the mRNA expression level of p16 was reduced in all experimental groups (the ornithine intake group (labeled "Aged+O" in the figure), the ornithine + sodium benzoate intake group (labeled "Aged+OB" in the figure), and the lactulose intake group (labeled "Aged+L" in the figure)) compared to old mice that had not been administered anything (labeled "Aged" in the figure) (Figure 3).

[0056] (2-3-4) Expression in skeletal muscle When the mRNA expression level of p16, a marker of aging cells, was measured in skeletal muscle, the mRNA expression level of p16 was reduced, although not significantly, in all experimental groups (the ornithine intake group (labeled "Aged+O" in the figure), the ornithine + sodium benzoate intake group (labeled "Aged+OB" in the figure), and the lactulose intake group (labeled "Aged+L" in the figure)) compared to old mice that had not been administered anything (labeled "Aged" in the figure) (Figure 4). [Industrial Applicability]

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

Claims

1. An agent for removing senescent cells, comprising one or more substances having an effect of promoting ammonia excretion in the body, the substances being one or more substances selected from the group consisting of ornithine, citrulline, taurine, carnitine, arginine, branched-chain amino acids (BCAAs), acetylglutamic acid, sodium benzoate, sodium phenylbutyrate, sodium phenylacetate, carglumic acid, lactulose, lactitol, and rifaximin.

2. 2. The senescent cell removing agent according to claim 1, wherein the substance having an ammonia excretion promoting effect in the body is ornithine, sodium benzoate, lactulose, or any combination thereof.

3. A senescent cell remover for preventing aging, comprising one or more substances having an effect of promoting ammonia excretion in the body, the substances being one or more substances selected from the group consisting of ornithine, citrulline, taurine, carnitine, arginine, branched-chain amino acids (BCAAs), acetylglutamic acid, sodium benzoate, sodium phenylbutyrate, sodium phenylacetate, carglumic acid, lactulose, lactitol, and rifaximin.

4. 4. The senescent cell removing agent for preventing aging according to claim 3, wherein the substance having an ammonia excretion promoting effect in the body is ornithine, sodium benzoate, lactulose, or any combination thereof.

5. A composition for removing senescent cells, comprising one or more substances having an effect of promoting ammonia excretion in the body, the substances being one or more substances selected from the group consisting of ornithine, citrulline, taurine, carnitine, arginine, branched-chain amino acids (BCAAs), acetylglutamic acid, sodium benzoate, sodium phenylbutyrate, sodium phenylacetate, carglumic acid, lactulose, lactitol, and rifaximin.

6. The composition for removing senescent cells according to claim 5, which is for pharmaceutical use.

7. 7. The composition for removing senescent cells according to claim 5 or 6, wherein the substance having an effect of promoting ammonia excretion in the body is ornithine, sodium benzoate, lactulose, or any combination thereof.

8. A composition for removing senescent cells to prevent aging, comprising one or more substances having an effect of promoting ammonia excretion in the body, the substances being one or more substances selected from the group consisting of ornithine, citrulline, taurine, carnitine, arginine, branched-chain amino acids (BCAAs), acetylglutamic acid, sodium benzoate, sodium phenylbutyrate, sodium phenylacetate, carglumic acid, lactulose, lactitol, and rifaximin.

9. The composition for removing senescent cells to prevent aging according to claim 8, which is for pharmaceutical use.

10. 10. The composition for removing senescent cells to prevent aging according to claim 8 or 9, wherein the substance having an effect of promoting ammonia excretion in the body is ornithine, sodium benzoate, lactulose, or any combination thereof.

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