JPWO2009066562A1 - Erythrocyte stem cell differentiation promoter and / or proliferation promoter, and use of methionine for preventing or treating senile anemia and a composition containing methionine - Google Patents

Abstract

Since anemia is frequently observed with aging, the first task is to develop a therapeutic agent effective in preventing senile anemia. Furthermore, since aging will progress in the future, it is a second object to provide an inexpensive and effective anti-aging agent with low side effects. It is a third object to provide a therapeutic agent effective for anemia caused by bone marrow function suppression, specifically, anemia as a side effect of administration of an anticancer drug. The inventor of the present application includes a simple substance of methionine (Met), an amino acid composition composed of one or more selected from methionine (Met), glycine (Gly), serine (Ser), and threonine (Thr). Ile, Trp, Thr, Val, His, Phe, Lys and Leu, (2) Ile, Thr, Val, His, Lys and Leu, (3) Trp, Thr, His, Phe and Lys, (4) Ile, An agent containing an amino acid mixture selected from Trp, Thr, Val, Phe, and Leu as an active ingredient has been found for the first time to have the ability to improve and recover senile anemia. Agent and cell antiaging agent could be provided.

Description

  The present invention relates to a technical field of erythroid stem cell differentiation promoter and / or proliferation promoter and anti-aging. The present invention relates to a technique for suppressing a decrease in function of erythroid stem cells such as hematopoietic stem cells and preventing aging, particularly in the proliferation of erythrocytes.

  With aging and aging, not only changes that appear on the surface of the body, such as wrinkles on the skin, hair loss, and white hair, but the function of the body gradually declines and various symptoms appear. Examples include diseases such as hypertension, myocardial infarction and angina, increased risk of diabetes due to decreased insulin production, osteoporosis, senile cataract and senile anemia.

  As a countermeasure against aging, various health foods and therapeutic drugs have been developed including folk remedies.

  For example, since melatonin decreases with aging, administration of melatonin to neurodegenerative diseases caused by aging has been studied, and in recent years, melatonin or its related substance (US Pat. No. 5,403,851) is said to have an antiaging effect. . Further, the action of melatonin is mediated by HGH, and growth hormone is also used as an anti-aging agent (Annu Rev Med. 2003; 54: 513-33. Epub 2001 Dec 3).

  In addition, active oxygen generated in the body causes damage to cells and DNA, lipid peroxidation, etc., and there are various effects due to involvement in many diseases, and the use of antioxidants to contain active oxygen is studied For example, vitamin C, vitamin E and the like are used, use of α-tocopherol and its analog (JP-A-8-277282), and radical scavengers have been developed (JP-A-9-241636). issue).

  Furthermore, among the symptoms associated with aging, there is a report that “active oxygen” generated in the body induces anemia and immunodeficiency in relation to anemia (Nature 431, 997-1002).

For senile anemia, as herbal medicine, for example, ginseng (carrot), 蒼朮 (jujutsu) or birches (bakujutsu), 茯苓 (bakuryo), licorice (licorice), ginger (shoyu), daikon (taiso), acid 棗Kami Kaishou-yu consisting of Jin (Sangoonin), Longan (Longan), Toshi (Onji), Toki (Otogi), Twilight (Ogi), Mika (Mokkou), Saiko (Psycho) and Shishi (Shishi) It has been known.
US Pat. No. 5,403,851 JP-A-8-277282 JP-A-9-241737 Annu Rev Med. 2003, 54, 513-33. Epub 2001 Dec 3 Nature 431, 997-1002

  Since anemia is frequently observed with aging, the first task is to develop a therapeutic agent effective in preventing senile anemia.

  In the future, the aging process will progress further, and it is a second problem to provide an inexpensive and effective anti-aging agent with low side effects.

  In addition, a third problem is to develop a therapeutic agent effective in preventing anemia that can be widely applied not only to the elderly.

  Furthermore, a fourth problem is to efficiently prepare red blood cells by cell culture for artificial blood production and the like.

  The inventor of this application has found that methionine (Met) and a methionine-containing amino acid composition have the effect of maintaining and promoting the hematopoietic function of erythrocytes, and are effective in improving senile anemia. Specifically, as an amino acid composition containing methionine, for the first time, it was found that an amino acid composition containing glycine (Gly) and glutamic acid (Glu) as an active ingredient in addition to methionine has the ability to improve and recover senile anemia. Is.

  The present invention also provides an efficient method for the proliferation and / or differentiation of red blood cells in vitro.

  The senile anemia prevention agent or senile anemia therapeutic agent containing methionine of the present invention as an active ingredient enables safe and inexpensive treatment for senile anemia, which is expected to increase in the future. Furthermore, the differentiation promoting agent and / or proliferation promoting agent of the erythroid stem cell of the present invention treats a wide range of anemias that require hematopoiesis such as anemia of intense athletes, side effects of anticancer agents and anemia due to renal diseases. Can improve. Furthermore, according to the present invention, it is possible to prevent a decrease in the function of cells accompanying aging, and to generally prevent the aging phenomenon. Further, according to the present invention, efficient proliferation and differentiation into red blood cells by cell culture becomes possible.

  This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2007-300814, which is the basis of the priority of the present application.

The hemoglobin amount in the peripheral blood of each group of mice immediately before the start of administration of the amino acid mixture. Mean ± standard deviation is shown. *: P <0.0001 (student ’s t-test for young mice) The amount of hemoglobin in the peripheral blood of each group of mice after administration of the amino acid mixture. Mean ± standard deviation is shown. *: P <0.01 (student ’s t-test for old mice (casein)) Effect of amino acid mixture on the growth of 707 fl. Cells. Mean ± standard deviation is shown. *: P <0.05 (student ’s t-test with no amino acid mixture added) Effect of 707 fl. Cell growth promotion effect when a specific amino acid is removed from an essential amino acid mixture. Mean ± standard deviation is shown. *: P <0.005 (student ’s t-test with no amino acid mixture added) Effect of methionine and threonine on 707fl. Cell proliferation. Mean ± standard deviation is shown. *: P <0.05 (student ’s t-test with no amino acid mixture added) The growth promoting effect of the amino acid mixture on human-derived K562 cells. Mean ± standard deviation is shown. *: P <0.05 (student ’s t-test with no amino acid mixture added) The growth promotion effect with respect to the human origin HEL92.1.7 cell of an amino acid mixture. Mean ± standard deviation is shown. *: P <0.05 (student ’s t-test with no amino acid mixture added) Effect of methionine concentration on 707 fl. Cell proliferation. Mean value ± standard deviation is shown. Mitigation effect of amino acid [threonine (Thr), or a mixture of glycine (Gly) and glutamic acid (Glu)] on the growth inhibitory activity of 707fl. Cells by high concentration methionine. Mean ± standard deviation is shown. *: P <0.05 (student t-test for control) Effect of methionine in anticancer drug actinomycin D-induced anemia model and transition of peripheral blood hemoglobin during the test period. Mean ± standard error is shown. Efficacy of methionine in the anticancer drug actinomycin D-induced anemia model. Peripheral hemoglobin level 17 days after the start of actinomycin D administration. Mean ± standard error is shown. Significant difference between different characters, P <0.05 (one-way ANOVA with Newman-Keuls's post hoc analysis) Methionine concentration and 707 fl. Cell proliferation. Mean ± standard deviation is shown. Statistical analysis is based on student ’s t-test. Changes in the expression level of erythrocyte-related genes due to differences in methionine concentration. Mean ± standard deviation is shown. Statistical analysis is based on student ’s t-test. Effect-1 of change in methionine concentration over time on expression level of erythroid differentiation-related gene. The medium used for the first 3 days, the next 24 hours, and the subsequent 24 hours of culture is a medium containing methionine at the next concentration. From the left in the figure: 1:30 μM → 30 μM → 30 μM 2: 30 μM → 150 μM → 150 μM 3: 30 μM → 150 μM → 30 μM 4: 30 μM → 150 μM →> 3 μM 5:30 μM → 150 μM + 1% DMSO → 150 μM + 1% DMSO average ± standard deviation is shown. *: P <0.05 (student ’s t-test for 2) Effect-2 of change in methionine concentration over time on expression level of erythroid differentiation-related gene-2. The medium used for the first 3 days, the next 24 hours, and the subsequent 24 hours of culture is a medium containing methionine at the next concentration. From the left in the figure: 1:30 μM → 30 μM → 30 μM 2: 30 μM → 150 μM → 150 μM 3: 30 μM → 150 μM → 30 μM 4: 30 μM → 150 μM →> 3 μM 5:30 μM → 150 μM + 1% DMSO → 150 μM + 1% DMSO average ± standard deviation is shown. *: P <0.05 (student ’s t-test for 2)

1. Introduction Anemia is simply a symptom, and is caused directly by reduced production of erythrocytes, decreased synthesis of hemoglobin, increased destruction of erythrocytes, and the causes are different.

  For example, the decrease in red blood cell production can be attributed to (i) decreased erythropoietin production due to renal failure, (b) abnormal hematopoietic stem cells seen in aplastic anemia, (c) bone marrow of leukemia or cancer, etc. ing. Moreover, the decrease in the synthesis of hemoglobin is cited as a cause of lack of iron, and treatment according to each cause is necessary.

2. Senile anemia and erythroid stem cell proliferation and erythroid stem cell to erythrocyte differentiation (differentiation and proliferation of erythroid stem cells)
In juvenile anemia, iron deficiency is often the cause. The cause of anemia due to aging has not been fully elucidated, but in contrast to juvenile anemia, it decreases the number of hematopoietic stem cells associated with aging of the bone marrow, shortens the lifespan of produced red blood cells, and atrophy of the gastric mucosa. It is cited as a cause of a decrease in the amount of nutrient absorption that accompanies it. At present, causes other than visceral hemorrhage and nutritional deficiencies are thought to be due to a decrease in blood stem cell function due to aging, particularly a decrease in self-renewal ability and induction of differentiation into red blood cells.

  Red blood cells are derived from hematopoietic stem cells, erythroblast burst-forming cells (BFU-E: burst-forming-unit-erythroid), erythroblast colony-forming cells (CFU-E: colony-forming-unit-erythroid), pro-red It is thought to be produced by differentiation from blasts, erythroblasts, reticulocytes and erythrocytes.

  The erythroid stem cells referred to in the present invention include hematopoietic stem cells, erythroblast burst-forming cells (BFU-E), and erythroid colony-forming cells (CFU-E). erythroid), proerythroblasts, and erythroid progenitors such as erythroblasts.

3. Due to prevention or treatment of methionine (Met) and senile anemia, suppression of cellular aging, promotion of differentiation and / or proliferation of erythroid stem cells, decrease in differentiation and / or proliferation function of erythroid stem cells accompanying aging, and side effects of anticancer agents Prevention or improvement of anemia caused by suppression of bone marrow function (hereinafter, amino acids may be indicated by a three-letter code)
3-1. The inventor of the present application experimentally found that an amino acid composition containing Met and Met has an effect of maintaining or promoting the hematopoietic function of erythrocytes and is effective in improving senile anemia.

  In addition, the administration of an amino acid composition containing Met and Met restores the expression level of a gene whose expression level decreases or increases with aging, so that the amino acid composition of this specific composition can be It has been found that it can be used as an agent for suppressing the decrease or malfunction of the cell, in other words, as a cell anti-aging agent.

  3-2. Furthermore, it has been found that an amino acid composition containing Met and Met promotes the growth of 707fl. Cells, which are erythroblast-like cells derived from the spleen of mice. At the same time, as a result of analyzing the gene expression of 707fl. Cells, which are erythroblast-like cells whose proliferation was promoted by Met, the expression of heme synthase and erythrocyte structural protein genes increased as in the case of old mice, Since the expression of genes such as interleukin (IL) mainly expressed in leukocytes decreased, it was estimated that differentiation into erythrocytes was promoted. At this time, the expression of genes related to epigenetic changes such as DNA (cytosine-5) -methyltransferase 1 gene (Dnmt1) and Histone deacetylase (Hdac) fluctuated, and Met promoted the growth of 707fl. Cells. Was expected to be accompanied by epigenetic changes in the cells. When Zebralin, an inhibitor of DNA (cytosine-5) -methyltransferase 1 (DNMT1), was actually allowed to act, Met proliferation-promoting activity on 707fl. Cells was inhibited.

3-3. Prevention and improvement of anemia caused by promotion of proliferation and / or differentiation of erythroid stem cells, reduction of proliferation and / or differentiation function of erythroid stem cells accompanying aging, and suppression of bone marrow function due to side effects of anticancer agents
It was found that Met promotes the proliferation of erythroid stem cells and the differentiation of erythroid stem cells into erythrocytes.

  In other words, through the differentiation and / or proliferation-promoting action of Met's erythroid stem cells, a wide range that requires hematopoiesis such as anemia of healthy individuals including the elderly, anemia of intense exercisers, side effects of anticancer drugs and anemia due to kidney disease By providing Met or a person with an amino acid composition containing Met, a decrease in hematopoietic function such as anemia can be prevented or improved.

  In addition, as an anticancer agent that causes anemia as a side effect, any anticancer agent that causes anemia as a side effect is targeted, and in particular, an anticancer agent that causes anemia due to suppression of bone marrow function can be mentioned. Examples thereof include platinum-based anticancer agents such as cisplatin and carboplatin, anthracyclines such as doxorubicin and epirubicin, and taxanes such as taxol and taxotere.

In the system using cultured cells, the present inventors have found that Met has an effect on the proliferation and differentiation of erythroblast-like cells as described above, and then the effect of having an effective concentration range and concentration of Met. Was examined. First, the effective concentration range of Met for promoting the growth of erythroblast-like cells is 25 μM to 50000 μM, preferably 30 μM to 5000 μM, more preferably 50 μM to 3500 μM, and even more preferably 50 μM to 200 μM. Can be mentioned. Note that there may be some mechanism that weakens the effect on cell proliferation when the Met concentration is in a certain range, higher than about 200 μM and below 2 mM. Alternatively, there is a possibility that there is a proliferative action in which the mechanism of cell proliferation differs depending on the concentration of methionine.

  It was also found that when the Met concentration is less than 25 μM, the proliferation effect on erythroblast-like cells is weak, but the differentiation promoting action on erythroblast-like cells is dominant.

  Further, it was found that the differentiation promoting action that predominates in a low concentration state of Met can be further enhanced by temporarily increasing the Met concentration and then decreasing the Met concentration again. That is, by giving a rapid change in Met concentration, the proliferation and differentiation of erythroid stem cells can be controlled more efficiently. When humans or animals ingest Met, it is expected that the Met concentration in hematopoietic tissues will rise transiently, and the proliferation of erythroid stem cells is thought to be promoted. Furthermore, since differentiation proceeds when the Met concentration decreases again, more red blood cells are produced as much as the original erythroid stem cells proliferate, and as a result, the number of red blood cells is considered to increase. It has been known for some time that hemolytic anemia is caused by excessive Met intake, which means that excessive Met intake continuously promotes the proliferation of erythroid progenitor cells and at the same time inhibits differentiation. As a result, it can be considered to induce hemolytic anemia because a large amount of undifferentiated red blood cells are produced.

4). Amino acid composition containing Met (amino acid preparation)
As an amino acid composition used in the present invention, as an active ingredient, an erythroid stem cell differentiation promoter and / or proliferation promoter, an anemia inhibitor, an anemia treatment agent, an senile anemia inhibitor, an elderly man, which contains an amino acid composition containing Met as an active ingredient A therapeutic agent for congenital anemia, or a cell anti-aging agent.

  The invention of the present application preferably contains, in addition to Met, an amino acid composition to which Gly and Glu are added as an active ingredient, an erythroid stem cell differentiation promoter and / or proliferation promoter, a senile anemia inhibitor, An anemia treatment agent or a cell antiaging agent is included.

  In the present invention, as an optional additive component to the amino acid composition, isoleucine (IIe), tryptophan (Trp), valine (Val), histidine (His), phenylalanine (Phe), lysine (Lys) and / or leucine ( Leu), an erythroid stem cell differentiation promoter and / or proliferation promoter, anemia inhibitor, anemia therapeutic agent, senile anemia inhibitor, senile anemia therapeutic agent, or cell Includes anti-aging agents.

  Specifically, an erythrocyte whose amino acid composition contains as an active ingredient an amino acid composition comprising Met, Gly, serine (Ser), threonine (Thr), Ile, Trp, Val, His, Phe, Lys and / or Leu A stem cell differentiation promoter and / or proliferation promoter, anemia prevention agent, anemia treatment agent, senile anemia prevention agent, senile anemia treatment agent, or cell aging prevention agent are included.

  Each amino acid constituting the amino acid composition of the present invention may be either D-form or L-form, preferably L-form. Further, it may be a free amino acid, or a pharmaceutically or food acceptable salt. For example, each amino acid may be hydrochloride, lactate, and the like.

  Examples of the present amino acid composition include the following.

4-1. In addition to Met, an erythroid stem cell differentiation promoter and / or proliferation promoter containing an amino acid composition to which one or more amino acids selected from the group consisting of Gly, Glu, Ser and Thr are added as an active ingredient, anemia An inhibitor, an anemia treatment agent, an senile anemia prevention agent, an senile anemia treatment agent, or a cell aging prevention agent.

  The present invention includes an agent for promoting differentiation and / or proliferation of an erythroid stem cell comprising an amino acid composition in which one or more amino acids selected from the group consisting of Gly, Glu, Ser and Thr are added to Met, and an anemia inhibitor An anemia treatment agent, a senile anemia prevention agent, a senile anemia treatment agent, or a cell aging prevention agent. The combined use of Gly and Glu shows an inhibitory effect on hemolytic anemia when Met is excessively consumed.

  Furthermore, the present invention includes an agent for promoting differentiation and / or proliferation of an erythroid stem cell comprising an amino acid composition in which Gly and Glu are added to Met, an anemia prevention agent, an anemia treatment agent, an senile anemia prevention agent, Therapeutic anemia or cell anti-aging agent is included. When Gly and Glu are used in combination and added to Met, the cytotoxic effect against high concentrations of Met can be mitigated, compared to adding Gly alone.

4-2. In addition to Met, an erythroid stem cell differentiation promoter comprising as an active ingredient an amino acid composition to which one or more amino acids selected from the group consisting of Ile, Trp, Thr, Val, His, Phe, Lys and Leu are added And / or a growth promoter, a senile anemia inhibitor, a senile anemia therapeutic agent, or a cell aging inhibitor.

  Preferably, (1) an amino acid mixture consisting of Ile, Thr, Val, His, Met, Lys and Leu, (2) an amino acid mixture consisting of Trp, Thr, His, Phe, Met and Lys, (3) Ile, Differentiation promoter and / or proliferation promoter of erythroid stem cells consisting of amino acid mixture selected from amino acid mixture consisting of Trp, Thr, Val, Phe, Met and Leu, anemia prevention agent, anemia treatment agent, senile anemia prevention Agent, senile anemia treatment agent, or cell antiaging agent.

5. Form of use of the composition of the present invention
Although the usage form of the amino acid composition of the present invention is not particularly limited, it can be used for food and drink, for nutritional supplement and for medical use. In the case of edible use, the amino acid composition of the present application can be edible as it is, but can also be used as an additive to various foods. In addition, the composition of the present invention can be dissolved in water to make a beverage. In that case, other nutritional components such as water-soluble vitamins and taurine can be further added. Moreover, in order to improve palatability, it can also be set as a drink by adding salts, such as sodium chloride, acids, such as a citric acid, and / or other suitable flavors. A pH adjusting agent and a chelating agent can be further added for stability.

  In the case of medical use, it can be used by general administration routes such as oral administration, tube administration, rectal administration, injection, administration by infusion. For oral administration, it can be mixed with the above composition itself or a pharmaceutically acceptable carrier, excipient, diluent, etc. to form powders, granules, tablets, capsules, troches, syrups, etc. . As an injection, an appropriate buffer, an isotonic agent or the like added and dissolved in sterilized distilled water may be used. Preferably, it can be administered orally.

  Since the Met and Met-containing amino acid composition of the present invention are extremely safe, the dosage can be set in a very wide range, and further varies depending on the administration method and purpose of use. For example, it can be orally administered in a range of 1 ml to 600 ml per day in a solution having a Met content of 0.1% to 1%. For example, when using as a composition which added Gly and Glu to Met, the ratio of Met: Gly: Glu can be used at 10: 1-10: 1-10, for example. The composition of the present invention can be expected to further enhance the effect when used in combination with iron, folic acid, vitamin B12 and the like.

  Specifically, for example, a range of about 10 mg / 50 kg body / day to 100 g / 50 kg body / day, preferably a range of about 100 mg / 50 kg body / day to 10 g / 50 kg body / day, more preferably The dosage is about 130 mg / 50 kg body / day. An effective Met concentration range for promoting the proliferation of erythroid stem cells is 25 μM to 50000 μM, preferably 30 μM to 5000 μM, more preferably 50 μM to 3500 μM, and even more preferably 50 μM to 200 μM. it can. The effective concentration range of Met when mainly differentiating erythroblasts into erythrocytes is less than 25 μM, preferably 0.01 μM to 20 μM, more preferably 0.1 μM to 10 μM, and even more preferably 0.1 μM to A range of 5 μM can be mentioned.

6). Culturing of erythroid stem cells 6-1. Cell culture additive The present invention further includes a cell culture medium additive for promoting proliferation or / and differentiation of erythroid stem cells, containing Met as an active ingredient.

  The proliferation promoter and / or differentiation promoter for erythroid stem cells containing Met of the present invention as an active ingredient are erythroid stem cells, that is, hematopoietic stem cells, erythroblast burst-forming cells (BFU-E). ), Erythroblast colony-forming cells (CFU-E: colony-forming-unit-erythroid), pre-erythroblasts, and erythroid progenitor cells such as erythroblasts can be added to the cell culture medium. As the cell culture medium for these erythroid stem cells, any medium may be used, and examples thereof include a commercially available medium for monopotential erythroid progenitor cells.

As the concentration added to the medium, first, the Met concentration range effective for promoting the proliferation of erythroid stem cells is 25 μM to 50000 μM, preferably 30 μM to 5000 μM, more preferably 50 μM to 3500 μM, and more preferably Can include a range of 50 μM to 200 μM. The effective Met concentration range for promoting the differentiation of erythroid stem cells is less than 25 μM, preferably 0.01 μM to 20 μM, more preferably 0.1 μM to 10 μM, and even more preferably 0.1 μM to 5 μM. 6-2. Method of culturing erythroid stem cells The present invention comprises (1) a step of growing erythroid stem cells using a medium containing Met at a high concentration, and (2) a step of differentiating using a medium containing Met at a low concentration. And a method of culturing erythroid stem cells. Furthermore, the present invention includes (1) a step of proliferating erythroid stem cells using a medium containing Met at a high concentration, and (2) erythrocyte stem cells proliferated in the step (1) again with a low concentration of Met. A method of culturing erythroid stem cells, comprising a step of further enhancing the differentiation promoting action by the step of differentiating using the medium contained in 1. The present invention also includes a method for culturing erythroid stem cells, comprising a step of efficiently controlling the proliferation and differentiation of erythroid stem cells by using a combination of mediums containing Met at low and high concentrations.

  As described above, the erythroid stem cells include hematopoietic stem cells, erythroblast burst-forming cells (BFU-E), and erythroid colony-forming cells (CFU-E). -unit-erythroid), proerythroblasts, and erythroid progenitors such as erythroblasts.

  The concentration of high-concentration Met contained in the step (1) is 25 μM to 50000 μM, preferably 30 μM to 5000 μM, more preferably 50 μM to 3500 μM, and even more preferably 50 μM to 200 μM. be able to. The concentration of the low concentration of Met contained in the step (2) is less than 25 μM, preferably 0.01 μM to 20 μM, more preferably 0.1 μM to 10 μM, and even more preferably 0.1 μM to 5 μM. Can range

Amino acid intake increases peripheral blood hemoglobin in aged mice
ICR male mice were bred under free drinking water and MF diet (oriental yeast) up to 20 months of age and divided into 3 groups according to body weight (old mice). As a control, 5-week-old ICR male mice were used (young mice). As shown in Table 1, AIN-93M refined feed (casein content 14%) was used as the basic feed, and 2% of the casein contained in this basic feed was replaced with an amino acid composition. Feed was used. During the test period, feed was freely consumed.

In the group giving amino acids, as shown in Table 2, the group (aged mouse (AAM1)) administered with amino acid mixture 1 (AAM1) mixed with 16 kinds of amino acids, and amino acid mixture 2 (AAM2) centered on essential amino acids. ) Group (aged mice (AAM2)). As a control, a group (casein administration group) fed with AIN-93M purified feed was provided with one group each of a young mouse and an old mouse (casein).

  Blood was collected from the tail vein of each group of old mice and young mice, and the amount of hemoglobin in the peripheral blood was measured with hemoglobin B-Test Wako (Wako Pure Chemical Industries). At this time, young mice were 5 weeks old, and old mice were 20 months old in each group. And young mice have n = 9, and old mice have n = 15 in each group. Statistical analysis was performed using a student's t-test and a significant difference test for young mice.

  The results are shown in FIG. AAM1 in parentheses of an aged mouse indicates a group scheduled to administer a mixture of 17 types of amino acids, and AAM2 indicates a group scheduled to administer a mixture of 12 types of amino acids (see Table 1 for types of amino acids). Prior to this administration, the amount of hemoglobin was significantly decreased in the aged mice compared to the young mice, indicating a decrease in hematopoietic function.

  Thereafter, the old mice of each group were ingested with the amino acid mixture AAM1 or AAM2 shown in Table 1 for 2 months. Thereafter, blood was collected from the tail vein, and the amount of hemoglobin in the peripheral blood was measured in the same manner. At this time, similarly to the above, n = 9 for young mice and n = 15 for each group of old mice. In the statistical analysis, a significant difference test for old mice in the casein administration group was performed using student's t-test.

  The results are shown in FIG. Although the recovery of hemoglobin was not observed in the old mice that did not receive amino acids, the peripheral blood hemoglobin level of the old mice that received amino acids recovered to the same level as that of young mice. Thus, it was shown that ingestion of the amino acid mixture promotes the erythropoiesis function and effectively increases the amount of peripheral blood hemoglobin decreased with aging.

Effect of Promoting Cell Proliferation of Mouse Erythrocyte-like Cells (707fl. Cells) by Amino Acid Mixture In Example 1, in order to identify the main body of amino acids that increased the amount of peripheral blood hemoglobin decreased by aging, 707fl. Cells ( ECACC (European Collection of Cell Cultures standard cells) was purchased). 707fl. Cells are erythroblast-like cells derived from mouse spleen that can differentiate into erythrocytes. The 707 fl. Cells were cultured in a RPMI1640 medium (SIGMA) supplemented with fetal calf serum so that the final concentration was 5% (RPMI1640 medium containing 5% fetal calf serum). At assay starting at about 1 × 10 ⁴ cell / well, the cells were seeded in 96-well microplate were added thereto an amino acid mixture in various concentrations (0.5% to 0.001%). At that time, the pH of the amino acid mixture was adjusted to around neutral and added. After culturing the cells for 3 days in this state, cell proliferation was measured by the WST-1 assay. That is, 10 μl of Premix WST-1 reagent (Takara Bio) is added to each well, incubated for 1 to 4 hours in a carbon dioxide incubator, and WST-1 by mitochondrial dehydrogenase in living cells. The amount of formazan produced by the cleavage of was measured at A450nm (reference A655nm).

  First, AAM2, which is a mixture of 12 amino acids centered on essential amino acids, and AAM3, which is a mixture of 9 essential amino acids, were examined (see Table 2 for the types of amino acids).

  The results are shown in FIG. Both AAM2 and AAM3 were effective in promoting the growth of 707 fl. Cells. The above results are consistent with the results obtained using the aged mice in Example 1, and the evaluation system using 707fl. Cells is effective in estimating the effect of promoting amino acid proliferation on erythroid stem cells. It has been shown.

Identification of Amino Acids as the Main Body of Growth Promoting Activity against Erythroid Stem Cells In Example 2, a mixture obtained by removing 2 to 3 amino acids from AAM3, an essential amino acid mixture in which growth promoting activity against erythroblast-like cells was observed It was dissolved in RPMI1640 medium containing 5% fetal calf serum, which is a cell culture medium. Here, the combinations when excluding amino acids were branched chain amino acids (Val, Leu, Ile), basic amino acids (His, Lys), aromatic amino acids (Trp, Phe), and other amino acids (Met, Thr). . At this time, the concentration of each amino acid was made equal to the concentration contained in the basic 1% solution of AAM3. A series of 2-fold dilution of each amino acid solution was dispensed at 50 μl into a 96-well microplate. An appropriately diluted cell (about 1 × 10 ⁴ cell) was added at 50 μl, and cultured in a carbon dioxide incubator for 3 days. Thereafter, 10 μl of WST-1 reagent was added to each well, incubated for 1 to 4 hours in a carbon dioxide incubator, and A450 nm (reference A655 nm) was measured to confirm cell proliferation.

  The results are shown in FIG. The growth promoting activity against 707fl. Cells was confirmed in the range of 0.004% to 0.008% in most of AAM3 and the mixture obtained by removing each amino acid from AAM3. However, when Met and Thr were removed, cell growth promoting activity was not observed, and it was strongly suggested that the body of growth promoting activity against erythroblast-like cells might be Met or Thr.

  Therefore, the proliferation promoting activity of Met and Thr against erythroblast-like cells was further examined by the same method as described above.

  The results are shown in FIG. Met showed growth promoting activity against erythroblast-like cells, but Thr did not show growth promoting activity, indicating that most of the growth promoting activity was dependent on Met. In addition, the optimum concentration of Met to promote the proliferation of erythroblast-like cells was estimated to be about 1 to 5 μg / ml. On the other hand, the growth promoting activity of AAM3 on erythroblast-like cells was observed around 0.005% (W / V), and the amount of Met contained at this time was 3.3% (W / V) = 1.65 μg / ml, which is almost in agreement with the current result.

  Here, it has been reported that excessive intake of Met causes hemolytic anemia as a side effect. It has also been reported that this phenomenon is suppressed by amino acids such as Gly, Ser, and Thr. Based on the above phenomenon and the effect of Met on cell proliferation promotion on erythroblast-like cells, when Met is ingested excessively, the proliferation of erythroid stem cells in hematopoietic tissues is excessively promoted and incomplete red blood cells are produced. It is estimated that hemolysis is promoted and anemia is induced.

The reason why hemolytic anemia induced by Met overdose is suppressed by amino acids such as Gly is thought to be because the addition of amino acids required as a material for biological components is effective as an auxiliary requirement in hematopoiesis . In other words, glutathione required for erythrocytes, Gly and succinyl CoA, which are the components of heme in erythrocytes, and branched chain amino acids, which are the components of succinyl CoA, in the induction of Met cell proliferation. (BCAA), Met, Gly synthetic material Ser, and succinyl CoA are also supplied from the citric acid cycle, so the addition of amino acids as the material of the biological components involved in the citric acid cycle This is considered to be an auxiliary requirement. Involvement of amino acids is considered not only to be a material of a biological component for erythrocyte synthesis but also to have an effect due to, for example, a quantitative balance between Gly and succinyl CoA. Gly is also considered to be a material for the synthesis of nucleic acids necessary for cell division and the biosynthesis of glutathione and creatine.

Amino acid growth-promoting effect on human-derived hematopoietic cells The effect of Met on cell proliferation was also examined in human cells. As human cells, K562 cells derived from chronic myeloid leukemia (erythroleukemia) and HEL92.1.7 cells derived from erythroblastic leukemia (both purchased cells from ECACC (European Collection of Cell Cultures) standard strain). Was used. Both cells have been reported to be differentiated into erythrocytes. At the start of the assay, cells were seeded in a 96-well microplate at about 1 × 10 ⁴ cells / well, and the amino acid mixture was added thereto at various concentrations (about 0.5% to 0.008%). At that time, the pH of the amino acid mixture was adjusted to around neutral and added. After culturing in this state for 3 to 4 days, cell proliferation was confirmed by WST-1 assay.

  The results are shown in FIGS. Similarly to 707fl. Cells, the proliferation promoting effect by the amino acid mixture was also observed for K562 cells and HEL92.1.7 cells. Further, AAM3, which is an essential amino acid mixture, was found to have a higher promoting effect on cell proliferation than AAM2. From the above study, it was suggested that essential amino acids have a promoting effect on erythropoiesis in humans as in mice.

707fl. Met amount required for cell growth
The effect of methionine concentration required for cell growth was examined.

Fetal bovine serum was added at a final concentration of 5% (Met-free RPMI1640 medium containing 5% fetal calf serum) to a medium (Met-free RPMI1640 medium) (Funakoshi) obtained by removing methionine from RPMI1640, which is a basic medium. A 100 mM Met solution was prepared in 5% fetal bovine serum-containing Met-free RPMI1640 medium. A 2-fold dilution series of the above Met solution was prepared with 5% fetal bovine serum-containing Met-free RPMI1640 medium, and dispensed into a 96-well microplate at 50 μl / well. Further, 707 fl. Cells were suspended in 5% fetal bovine serum-containing Met-free RPMI1640 medium at 2 × 10 ⁵ cells / ml, and added to wells containing Met dilution series at 50 μl / well. After culturing for 3 days in a carbon dioxide incubator, the degree of cell proliferation was confirmed by the WST-1 assay.

  The results are shown in FIG. When the Met concentration was 25 μM to 50000 μM, a cell growth promoting effect of 707 fl. Cells was observed, and the highest cell growth promoting effect was observed at a concentration of 50 μM to 200 μM.

Examination of amino acids that mitigate cell growth inhibition by high concentration Met As shown in Example 3, high concentration Met inhibits the growth of 707 fl. Cells compared to the case of Met concentration optimal for cell growth. Thus, amino acids that mitigate the growth inhibitory activity caused by high concentrations of Met were examined using mitigation as an index. As each amino acid to be used in the test, (i) Thr, or (b) Gly and Glu were used, which were each mixed with the same weight as Met. Each amino acid mixture was dissolved in 5% fetal calf serum-containing RPMI1640 medium, which is a cell culture medium, and 50 μl of the dilution series was dispensed into a 96-well microplate. 50 μl of appropriately diluted cells (approximately 5 × 10 ⁴ cells / 50 μl) were added thereto, and the cells were cultured in a carbon dioxide incubator for 3 days. After this, 10 μl of WST-1 reagent was added to each well, further incubated for 2-4 hours, A450 nm (reference A655 nm) was measured, and cell proliferation was compared.

  The results are shown in FIG. Cell growth inhibition was detected at a concentration of Met, 12.5 μg / ml and above. When Thr was present in the same concentration as Met, the inhibitory activity could be delayed to a range of 12.5 μg / ml or more and less than 25 μg / ml. On the other hand, when Gly and Glu were allowed to coexist with Met at the same concentration, suppression of the growth inhibitory effect exerted by 50 μg / ml or more of Met was observed, indicating that the effect was higher than Thr.

Efficacy of Met in the anticancer drug actinomycin D-induced anemia model [method]
The introduced mice were acclimated for 10 days. On the seventh day of acclimatization, 10 μl of blood was collected from the tail vein and the amount of hemoglobin (Hb) was measured. The body weight was also measured and divided into groups based on Hb amount and body weight. After grouping, the mice were given intraperitoneal administration of actinomycin D twice for 5 consecutive days with an interval of 2 days. The dose was 0.07 mg / kg body mass / day, which is half of the LD50 when administered intraperitoneally for 5 days in the mouse. Actinomycin D was dissolved in physiological saline and administered at 100 μl / body. Further, physiological saline, not actinomycin D, was intraperitoneally administered at a dose of 100 μl / body to serve as a negative control group.

  From the start of actinomycin D administration to the end of the test, an amino acid aqueous solution or sterilized water was forcibly administered orally. The concentration of the Met aqueous solution was 0.2%, 1%, and 5% aqueous solution. Sterile water was used as a negative control. A 1% Thr aqueous solution was used as a negative control for the amino acid aqueous solution related to the hematopoietic effect. The dosage was 200 μl / body / day for any amino acid aqueous solution or sterilized water.

During the test period, 10 μl of blood was collected from the tail vein at an appropriate frequency, and the amount of Hb was measured.

  After completion of the above test, the mice were subjected to whole blood collection under anesthesia and sacrificed, and spleen and bone marrow tissues were collected for gene analysis.

  The group composition (10 animals in each group) is as follows.

Group 1: Intraperitoneal saline, oral sterilized water group Group 2: Actinomycin D (0.07 mg / kg body mass / day) intraperitoneal, sterile water oral group 3: Actinomycin D ( 0.07mg / kg body mass / day) Intraperitoneal administration, 1% Thr aqueous solution oral administration group Group 4: Actinomycin D (0.07mg / kg body mass / day) intraperitoneal administration, 0.2% Met aqueous solution oral administration group 5 Group: Actinomycin D (0.07mg / kg body mass / day) intraperitoneal administration, 1.0% Met aqueous solution oral administration group Group 6: Actinomycin D (0.07mg / kg body mass / day) intraperitoneal administration, 5.0% Met Oral solution administration group [Result]
FIG. 10 shows the results of measuring the fluctuation of the peripheral blood Hb amount during the test period. The decrease in Hb amount reached the maximum 17 days after the start of administration. FIG. 11 shows the amount of peripheral blood Hb 17 days after the start of administration. At this time, the 1% Thr aqueous solution administration group was not statistically different from the sterilized water administration group. On the other hand, in the 0.2% Met aqueous solution administration group, an increasing tendency of Hb was observed, and in the 1% and 5% Met aqueous solution administration groups, there was a significant difference.

This study confirmed again that Met has the effect of suppressing or improving anemia associated with suppression of bone marrow function. In order to suppress or ameliorate anemia associated with bone marrow function suppression, oral ingestion at a Met concentration of 1% was found to be sufficiently effective, but an effect can also be expected at a Met concentration of 0.2%. When the dose of 1% Met is converted to human, it is approximately 130 mg / 50 kg body / day in terms of body surface area. This is an amount that can be sufficiently administered to humans. It was confirmed that Met's erythropoiesis-promoting effect was effective not only for recovering the amount of peripheral blood Hb decreased in old mice, but also for anemia caused by side effects of anticancer drugs.

Analysis of gene expression levels in spleen and bone marrow of Met-administered actinomycin D-induced anemia mice
Spleen and bone marrow tissues were collected from the mice provided in Example 7, immersed in an RNA rater reagent manufactured by Ambion about 10 times the volume of the tissues, and allowed to stand overnight at 4 ° C. The treated tissue was stored at −80 ° C. until RNA was extracted. RNA extraction and purification from the preserved tissue was performed using Qiagen RNeasy kit according to the attached manual. The quality of the RNA after purification was confirmed using an Agilent bioanalyzer. Further, DNA microarray analysis using purified RNA was performed using Affymetrix GeneChip, mouse 430_2.0 array. The effect of administration of 1% Met to mice in which anemia was induced by actinomycin D administration (in mice subjected to Example 7 group 5) was compared with that in the amino acid administration group in which anemia was induced by actinomycin D and sterilized water was administered. It examined by comparison of the gene expression level with the mouse | mouth used as a control | contrast (The mouse | mouth used for Example 7 2nd group).

〔result〕
Table 3 shows erythrocyte-related genes whose expression was increased by administration of 1% Met. Table 3 shows the relative expression levels relative to the expression levels of each gene in mice in which anemia was induced with actinomycin D and sterilized water was administered.

In both spleen and bone marrow, which are erythropoietic tissues of mice, many of the genes associated with erythrocytes were elevated compared to mice administered with sterilized water. As a result, it was shown that the administration of Met promotes the differentiation of erythrocytes. In addition, the up-regulated genes are c-kit (also known as CD117 transmembrane tyrosine kinase) expressed from bone marrow stem cells to CFU-E and expressed in hematopoietic progenitor cells such as colony-forming cells, but B cell lineage (The interaction between CD117 and ligand is important for hematopoiesis.) And its ligand (stem cell factor: also referred to as SCF). It extends to transferrin receptor (CD71), heme synthase and erythrocyte membrane protein. Therefore, it can be said that the effect of Met extends over a wide range from unipotent stem cells differentiated into erythrocytes to erythroblasts.

Relationship between Met concentration and erythroblast-like cell differentiation and proliferation promoting effect The concentration of Met in mouse or human plasma is about 30 μM. Furthermore, in the case of bone marrow, which is the main hematopoietic tissue, the Met concentration in the tissue fluid is below the detection limit (<3 μM). Therefore, the extracellular Met concentration in normal hematopoietic tissues is considered to be considerably lower than in vitro cell culture conditions. Moreover, from FIG. 8 mentioned above, it can be seen that Met shows a growth promoting effect at 25 μM to 50000 μM against erythroblast-like cells.

Therefore, we compared the gene expression of 707fl. Cells when Met was present at a concentration of 150 μM, which showed a growth promoting effect on erythroblast-like cells, and when there was almost no Met as in the case of bone marrow fluid.

〔Method〕
A medium in which fetal bovine serum was added to a Met-free RPMI1640 medium to a final concentration of 5% (5% fetal bovine serum-containing Met-free RPMI1640 medium) was prepared. Further, a medium (MRP containing 5% fetal bovine serum-containing RPMI1640 medium containing each concentration of Met) was prepared by adding Met to the above-mentioned medium to have each concentration.

707 fl. Cells were cultured in RPMI1640 medium containing 5% fetal bovine serum containing 30 μM Met for 3 days. The cultured cells were collected in two minutes, and one was resuspended in RPMI1640 medium containing 5% fetal bovine serum and not containing Met at a concentration of about 5 × 10 ⁵ cells / ml. Here, the 5% fetal bovine serum-containing Met-free RPMI1640 medium contains 5% fetal bovine serum and therefore contains about 1 to 3 μM of serum-derived Met. The other was resuspended in RPMI1640 medium containing 5% fetal calf serum containing 150 μM Met at a concentration of approximately 5 × 10 ⁵ cells / ml. Further, the cells under each condition were divided into 3 parts and cultured for 24 hours, respectively, and a portion of the cell culture solution was taken and the degree of cell proliferation was confirmed by the WST-1 assay.

〔result〕
Cells cultured in RPMI1640 medium containing 150% Met containing 5% fetal bovine serum (150µM Met) increased significantly compared to cells cultured in RPMI1640 medium containing no 5% fetal calf serum (> 3µM Met) Was observed (Figure 12). Furthermore, RNA was extracted from these cells, and the expression levels of erythrocyte-related marker genes were compared by real-time PCR. As the erythroid differentiation marker, the expression level of the gene for glycophorin A, a protein that is abundant and specifically present in the cell membrane of erythrocytes, and ferrochelatase, which is one of the rate-limiting enzymes of heme biosynthesis, was measured. At this time, beta actin was used as a reference gene. As a result, the cells cultured in 5% fetal bovine serum-containing Met-free RPMI1640 medium (> 3μM Met) were compared to the cells cultured in 150μM Met-containing 5% fetal bovine serum-containing RPMI1640 medium (150μM Met). Expression was significantly higher (Figure 13). Therefore, when Met is present at a higher concentration than normal, erythroid stem cells are more proliferating than differentiation, and conversely, when Met is changed to a lower concentration, differentiation is more promoted than proliferation. It has been shown. From the above results, it was considered that the erythroid stem cells have a sensor function that senses the Met concentration, and that proliferation and differentiation are skillfully controlled by the extracellular Met concentration.

Relationship between erythroblast-like cell differentiation and growth-promoting effect with changes in Met concentration [method]
Add 5% fetal bovine serum-containing Met-free RPMI1640 medium to a concentration of 150 μM, and then add dimethyl sulfoxide (DMSO) to a final concentration of 1% (containing 150 μMMet) 1% DMSO-containing RPMI1640 medium containing 1% fetal bovine serum) was prepared.

707 fl. Cells were divided into 5 groups from 1 to 5 in 5% fetal bovine serum-containing RPMI1640 medium (30 μM) containing 30 μM Met and cultured for 3 days. Thereafter, the first group was again added to 5% fetal bovine serum-containing RPMI1640 medium (30 μM) containing 30 μM Met, and the second group was added to 5% fetal bovine serum-containing RPMI1640 medium (150 μM) containing 150 μM Met. Each group was replaced with RPMI1640 medium (150 μM + 1% DMSO) containing 1% DMSO containing 5% fetal bovine serum containing 50 μM Met, and cultured for 24 hours.

Furthermore, the first and third groups are in RPMI1640 medium (30 μM) containing 5% fetal bovine serum containing 30 μM Met, the second group is RPMI1640 medium (150 μM) containing 5% fetal bovine serum containing 150 μM Met, and the fourth group. Replaced with 5% fetal bovine serum-containing Met-free RPMI1640 medium (> 3 μM) and the fifth group with 150 μM Met-containing 5% fetal bovine serum-containing 1% DMSO-containing RPMI1640 medium (150 μM + 1% DMSO). And cultured for 24 hours. Here 1% DMSO was added as a positive control for differentiation. After completion of the culture, RNA was extracted from each cell, and the gene expression levels of glycophorin A and ferrochelatase, which are erythrocyte-related marker genes, were compared by real-time PCR. In addition, beta actin was used as a reference gene at this time.

〔result〕
Compared with cells cultured continuously at a Met concentration of 30 μM, cells cultured at a Met concentration of 150 μM have decreased expression of erythroid differentiation-related genes, and it can be said that proliferation is promoted by differentiation. On the other hand, when the Met concentration is reduced to less than 3 μM, the expression of differentiation-related genes is greater than when culturing continuously at a Met concentration of 30 μM or by replacing the medium containing 150 μM Met with a medium containing 30 μM Met. The level increased and reached almost the same level as when cultured in the presence of 1% DMSO (FIGS. 14 and 15). These results indicated that the rapid induction of Met concentration induced efficient differentiation from cell proliferation to differentiation in 707fl. Cells, which are erythroblast-like cells. Therefore, Met has been shown to efficiently control the proliferation and differentiation of erythroid stem cells.

  The present invention provides a preventive agent for senile anemia and a therapeutic agent for senile anemia, and can be used in the pharmaceutical manufacturing industry or the food manufacturing industry.

  All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (10)

  An agent for promoting differentiation and / or proliferation of erythroid stem cells containing Met as an active ingredient.   Furthermore, the differentiation promoter and / or proliferation promoter of erythroid stem cells according to claim 1, further comprising Glu and Gly. Furthermore, the differentiation promoter and / or proliferation promoter of erythroid stem cells according to claim 1, further comprising an amino acid mixture selected from the following (1) to (3).
(1) Ile, Thr, Val, His, Lys, and Leu
(2) Trp, Thr, His, Phe, and Lys
(3) Ile, Trp, Thr, Val, Phe, and Leu   A food or drink containing the erythroid stem cell differentiation promoter and / or growth promoter according to any one of claims 1 to 3.   The anemia prevention agent or anemia therapeutic agent containing the differentiation promoter and / or proliferation promoter of the erythroid stem cell according to any one of claims 1 to 3.   A senile anemia prevention agent or a senile anemia treatment agent comprising the erythroid stem cell differentiation promoting agent and / or proliferation promoting agent according to any one of claims 1 to 3.   A cell aging inhibitor containing Met as an active ingredient.   The cell aging inhibitor according to claim 7, further comprising Glu and Gly. Furthermore, the cell aging inhibitor of Claim 7 containing the amino acid mixture selected from the following (1)-(3).
(1) Ile, Thr, Val, His, Lys, and Leu
(2) Trp, Thr, His, Phe, and Lys
(3) Ile, Trp, Thr, Val, Phe, and Leu   A food or drink containing the cell antiaging agent according to any one of claims 7 to 9.

Images