JPH09110686A - Macrophage nitrogen monoxide-producing sthenic agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a macrophage nitrogen monoxide-producing sthenic agent exhibiting immunoactivating action by exasperating NO-producing amount of a macrophage and having protecting and curing effect to infection, containing an amino acid composition mixed with a specific amount of arginine as an active component. SOLUTION: This macrophage nitrogen monoxide-producing sthenic agent contains an amino acid composition blended with 10-30wt.% arginine. Preferably, the composition satisfies following conditions (1)-(3): (1) fractions of essential amino acids to whole of amino acids in the composition are each: 4.0-8.0wt.% L-isoleucine; 7.0-14.0wt.% L-leucine; 5.0-10.0wt.% L-lysine; 3.0-6.0wt.% L- methionine and L-cystine; 6.0-9.0wt.% L-phenylalanine and L-tyrosine; 4.0-6.0wt.% L-threonine; 1.0-2.0wt.% L-tryptophan; 5.0-10.0wt.% L-valine; 2.0-4.0wt.% L- histidine; (2) a fraction of L-arginine to whole of amino acids in the composition is 10-30wt.%; and (3) a weight ratio of L-arginine to L-lysine in the composition is 1-4.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a nitric oxide production-enhancing agent for macrophages, which comprises, as an active ingredient, an amino acid composition containing 10 to 30% by weight of arginine based on all amino acids. The nitric oxide production-enhancing agent of the present invention enhances nitric oxide production in macrophages by administering it, enhances immune function, does not cause side effects, invasive period such as postoperative period, or immunodeficiency, It is useful for the prevention and treatment of infectious diseases caused by radiation therapy, administration of anticancer agents, acquired immunodeficiency, and the like.

[0002]

2. Description of the Related Art Conventionally, in determining the intake requirement of human amino acids, emphasis has been placed on balanced intake of essential amino acids, and non-essential amino acids (essential amino acids).
Was used as an index for the growth rate of animals by Rose and the maintenance of nitrogen equilibrium (Physiol. Rev., vol.18, pp.109-136).
(1938)), it has not been necessary. However, in recent years, it has been pointed out that the types and amounts of amino acids required for the living body to exhibit a physiologically optimal reaction are larger than those estimated by nitrogen equilibrium and the like. In particular, under various pathological conditions, for example, branched-chain amino acids (leucine, isoleucine, valine) in the invasive period (hereinafter, all amino acids not specified are L-type amino acids except glycine) and glutamine required amount. Increase.
And Seifter et al. Have shown that the intake of arginine shortens the wound healing days.
It is reported that arginine is supplied after being converted into proline, which is a component of collagen necessary for granulation, and also has an action of stimulating hormone secretion such as insulin and glucagon which have an effect on wound healing. Sur
gery, vol.84, pp.224-230 (1978)). Also, Barbul et al.
Enhancement of immune function was observed by administering 30 g of arginine to normal subjects (Surgery, vol.90, pp.244-251 (198
1)) Further, Reynolds et al. Also reported that arginine has an effect on immunity such as promotion of differentiation of cytotoxic T lymphocytes (Surgery, vol.104, pp.142-151). (19
88)). These facts suggest that arginine is not only a constituent of somatic proteins, but also necessary for maintaining physiological functions. Therefore, the amount of arginine required to maintain the body is not accurate according to the Rose standard,
It is considered to be even higher (Yoyoshi Wakabayashi et al., Surgery and metabolism and nutrition, vol.25, 8.398-404 (1991)). Therefore, if the amount of arginine synthesized in the body decreases due to disease, etc., or if the amount of arginine supplied in the nutritional management during the disease stage is less than the required amount, delay in healing and recovery from the disease, exacerbation of symptoms, complications, etc. Is afraid.

Further, in normal animals, a sufficient amount of arginine is not uniformly synthesized in all cells, but basically it is synthesized from glutamic acid by seven kinds of enzymes under the sharing of the major organs of the small intestine and the kidney. It is said that it is sent to various parts of the body and consumed in protein synthesis, urea cycle, and others (Yoyoshi Wakabayashi, Surgery and Metabolism and Nutrition, vol.25, pp.398-404 (1991)).
Therefore, patients with intestinal disorders such as mass resection of the small intestine and short bowel syndrome, patients with abnormally increased protein metabolism due to severe invasion such as trauma or burns, or intestinal disorders or intestinal tracts seen as side effects during anticancer drug administration In patients with atrophy, etc.,
Synthetic organ function of arginine is remarkably reduced, arginine is very likely to be essential, arginine deficiency is exhibited even in nutritional management with normal nutritional composition, and exacerbation of symptoms and delay of recovery, and further Is at risk of developing an infectious disease.

As the action of arginine under pathological conditions, in addition to the wound healing effect and immunostimulatory effect, a nitrogen saving effect, ammonia detoxification, endocrine system stimulation, creatine / polyamine synthesis and the like are known. And, as those utilizing these effects, for example, amino acid preparations for patients with liver failure that utilize ammonia detoxification (JP-A-1-83017), immunostimulatory compositions that utilize immunostimulatory action (JP-A-2 -191213), immunostimulants (Japanese Patent Publication No. 5-502881), amino acid formulations for cancer (Japanese Patent Publication No. 5-79049, Japanese Patent Publication No. 3-68514, Japanese Patent Publication No. 6-40900), etc. Is proposed. More recent studies show that L-arginine-
Nitric oxide produced from arginine by the nitric oxide pathway has a vasodilatory effect, and it was revealed that the production of nitric oxide was significantly increased with an increase in the dose of arginine. It has been found that it plays an important role in function regulation and cell-cell communication.
However, the nitric oxide produced from this arginine is
While exerting the above-mentioned immunostimulatory effect in the living body, since it is generally a radical gas, it also has toxic effects such as cell damage and carcinogenicity, and has two sides of good and bad for the living body.
In addition, oral or enteral overdose of arginine
It is known that diarrhea occurs via nitric oxide synthesis and cyclic GMP synthesis, and there is concern that diarrhea may impair the absorption of nutrients from the intestinal tract to reduce systemic nutritional status. That is, arginine during overdose is a “double-edged sword” substance under pathological conditions, and controlling these actions of arginine and ornithine, which is a derivative of arginine, has become an important therapeutic issue. .

On the other hand, the immune function is decreased during the postoperative invasive period, during radiation therapy and anticancer drug administration in cancer patients, and also during acquired immunodeficiency, and bacteria or attenuated bacteria considered to be normal harmless. Infectious diseases (opportunistic infections) are easily caused by bacteria, protozoa, etc. Examples of nutritional therapy for such patients include parenteral nutrition therapy such as infusion, enteral or oral therapy. Enteral or oral therapy has a more physiological route of administration than parenteral nutrition therapy such as infusion and is not only safer, but also has coagulation dysfunction, liver dysfunction, respiratory dysfunction, and reticuloendothelial system. It has attracted attention because of its features such as fat deposition, impaired immune function, and difficulty in causing complications such as fat embolism. In particular, since bacterial translocation due to intestinal atrophy that occurs during intravenous therapy is unlikely to occur in enteral therapy, the proportion of oral or enteral nutrition therapy in medical treatment is increasing. However, all of the compositions used for nutrition therapy up to now are intended only for the purpose of recovering and maintaining the nutritional status of patients, and nutritional compositions for the purpose of preventing and treating infectious diseases have not yet been developed. Not in the present situation.

[0006]

DISCLOSURE OF THE INVENTION The present invention focuses on the above-mentioned usefulness of arginine, in particular, on the immunostimulatory action involving nitric oxide, and prevents and treats the onset of infectious diseases when the immune function is lowered. As a result of diligently searching for a composition capable of causing no side effects such as diarrhea, by incorporating arginine in a certain range in the amino acid composition, there are no side effects and the amount of nitric oxide production by macrophages in the living body is increased. By doing so, they have found that the onset of infection can be prevented and treated. That is, the present invention is useful for enhancing nitric oxide production in macrophages useful for the prevention and treatment of infectious diseases that occur during the invasive period such as post-operative period, or immunodeficiency, radiation therapy, anticancer drug administration, acquired immunodeficiency, etc. It is an object to provide an agent.

[0007]

The present invention relates to a nitric oxide (NO) production enhancer for macrophages, which comprises as an active ingredient an amino acid composition containing 10 to 30% by weight of arginine based on all amino acids. Administration of the enhancer of the present invention enhances nitric oxide production in macrophages, and infections that occur during the invasive period such as post-operative period or with immunodeficiency, radiation therapy, anticancer drug administration, acquired immunodeficiency syndrome, etc. Pharmaceutical and nutritional compositions useful for the prevention and treatment of diseases are provided. In the present invention, the amino acid composition is prepared according to the following conditions. (1) The essential amino acid composition is set to a ratio close to the essential amino acid composition of milk protein such as human or bovine or egg protein such as egg white. The essential amino acid composition is shown in Table 1.

[0008]

[Table 1]

(2) Regarding the composition of non-essential amino acids, the ratio of arginine to the total amino acids is 10 to 30% by weight, preferably 20 to 25% by weight. (3) The non-essential amino acid composition excluding arginine is set to a ratio close to the non-essential amino acid composition of milk protein such as human and bovine, egg protein such as egg white, and soybean protein.
The non-essential amino acid composition is shown in Table 2.

[0010]

[Table 2]

By blending the amino acid composition of the active ingredient of the present invention as a nitrogen source of the nutritional composition according to the above conditions, arginine deficiency in the body due to protein metabolism increase is caused to a wide range of disease patients. It is possible to prevent or treat the infectious disease by promptly eliminating it, and in the state where side effects such as diarrhea and the like and amino acid imbalance in the body do not occur, the physiological action of arginine, particularly the immunostimulatory action is maximized.

The amino acid composition of the macrophage nitric oxide production-enhancing agent of the present invention includes free crystalline amino acid and a pharmacologically acceptable salt thereof, for example, a metal salt such as sodium salt and a hydrochloride salt. It is possible to use those in the form of organic acid salts such as mineral acid salts and acetate salts. Alternatively, a part or all of the free crystalline amino acid may be used in the form of N-acyl derivative, and a substance in which two kinds of amino acids are salt-bonded, for example, L-arginine.L-glutamate or L
-Lysine / L-aspartate may be used.
Furthermore, milk protein such as casein or whey protein, egg protein such as egg white, or a hydrolyzate thereof is used as a base, and free arginine or a synthetic peptide containing arginine, or arginine such as protamine, which is a fish algal protein. The arginine content of the protein may be adjusted by adding a protein containing a large amount of or, a hydrolyzate thereof, or the like. In particular, based on a nitrogen source or milk protein hydrolyzate or egg protein hydrolyzate consisting only of free crystalline amino acids,
When a composition (nitrogen source) in which free arginine or a peptide containing arginine is added to adjust the arginine content is used, absorption from the intestinal tract is rapid, and thus a better effect can be expected. Further, the nitric oxide production-enhancing agent of the macrophage of the present invention can be prepared by mixing the above-mentioned nitrogen source.
It is practically preferable to use nutrients such as lipids, vitamins and minerals as nutritional supplements containing necessary amounts of them.

The nitric oxide production-enhancing agent of the present invention is preferably administered by adjusting the concentration of nitrogen source to a solution of 2 to 12% by weight. When blending energy sources such as sugars and lipids, the ratio of the amount of calories derived from sugars and lipids to the amount of nitrogen derived from nitrogen sources (Non-protein Cal /
It is desirable to adjust the N ratio) so that it falls within the range of 90 to 200, and the concentration of sugars and lipids within the range of 0.5 to 2 kcal / ml before administration. The sugar that can be added to the nitric oxide production enhancer of the present invention is not particularly limited, and glucose, sucrose, fructose, dextrin, or the like may be used. In particular, when dextrin is used, the osmotic pressure can be kept low during oral enteral administration, and the occurrence of diarrhea resulting from high osmotic pressure during administration can be reduced. The lipid is not particularly limited, and soybean oil, safflower oil, corn oil, perilla oil, etc. may be used. Since it is desirable that the lipid serves as a supply source of linoleic acid which is an essential fatty acid, it is preferable to add soybean oil, safflower oil or the like containing linoleic acid. The amount of the linoleic acid is not particularly limited, but if linoleic acid is added in an amount of 2 to 4 calories with respect to the total calories, essential fatty acids are not deficient. It has recently been confirmed that it is another essential fatty acid.
α-linolenic acid, which is an n-3 polyunsaturated fatty acid, is preferably used together with linoleic acid in an α-linolenic acid / linoleic acid ratio of about 1/5 to 1/3. That is, α-
Addition of linolenic acid can prevent the occurrence of numbness, paralysis, muscle weakness, visual impairment, etc., which are clinically recognized as deficiency symptoms of n-3 polyunsaturated fatty acids.

The method for preparing the nitric oxide production enhancer of the present invention is substantially the same as the method for preparing an ordinary oral enteral nutrition composition, for example, by powder-mixing the raw materials containing the above-mentioned components. It can be prepared or dissolved to prepare a liquid preparation, or an appropriate emulsifier is added to prepare an emulsion liquid preparation. Furthermore, if necessary, a preservative such as erythorbic acid, a pH adjusting agent such as citric acid, malic acid, sodium carbonate, or the like, or other additives can be added. In addition, it is desirable that the liquid agent is sterilized by means such as heat sterilization or sterile filtration. In addition, it is desirable that the powder be dissolved or suspended in water or warm water immediately before administration so that it can be administered. The nitric oxide production enhancer of the present invention thus prepared is orally or enterally administered, and the dose may be the same as the dose of a usual oral enteral nutrition composition, Generally, the amount of nitrogen source for adults should be 20 to 120 g per day. Oral and enteral nutritional compositions containing saccharides, lipids, etc., which are prepared so that their content is 1 kcal / ml, 200 to 2,400 ml should be used as a guideline for the patient's condition, It may be appropriately increased or decreased according to nutritional status, age, weight, etc.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is described in more detail by the following examples, but the present invention is not limited to these.

[0016]

[Example 1] Production of nutritional composition of the present invention According to the blending tables of Table 3, Table 4 (blending of minerals) and Table 5 (blending of vitamins), the amino acid composition shown in the column of Example 1 of Table 6 The nitrogen source of was dissolved and suspended together with other raw materials, the pH was adjusted to near neutral with citric acid, and the mixture was emulsified with a high pressure homogenizer. Next, this emulsion was filled in a synthetic resin bag, the space was purged with nitrogen, sealed, and heat-sterilized by a conventional method to produce the target nitric oxide production enhancer of the macrophage of the present invention.

[0017]

[Table 3]

[0018]

[Table 4]

[0019]

[Table 5]

[0020]

[Table 6]

[0021]

Example 2 Measurement of Survival Rate by Intraperitoneal Infection of Listeria Regarding the effect of the nitric oxide production enhancer of the macrophage of the present invention, the survival prolongation effect by intraperitoneal administration of Listeria was tested. That is, the enhancer of the present invention and comparative nutritional compositions 1 and 2 (hereinafter referred to as Comparative Examples 1 and 2)
ICR mice weighing 25 to 30 g (13 mice per group) were allowed to freely ingest for 1 week, and then 4 x 10 ⁵ live Listeria (acquired from the Department of Bacteriology, Niigata University School of Medicine) were intraperitoneally injected to infect them. It was After that, the survival rate was calculated by observing life and death daily. In Comparative Examples 1 and 2, those produced by using the nitrogen source having the composition of each comparative example shown in Table 6 by the same method as the production of the enhancer of the present invention were used. Comparative Example 1 contains no arginine, and Comparative Example 2 contains 31% by weight of arginine based on the total amino acid composition. The results are shown in FIG.

As a result, the survival rate of the group that constantly ingested the enhancer of the present invention during the period was higher than that of Comparative Examples 1 and 2.

[0023]

Example 3 Measurement of Number of Bacteria in Organ Due to Infection of Listeria by Intravenous Administration The number of bacteria in an organ due to intravenous administration of Listeria was measured for the effect of the enhancer of the present invention. That is, ICR mice weighing 25 to 30 g (5 mice per group) were allowed to freely ingest each composition similar to that of Example 2 for 1 week, and then 4 × 10 ⁵ live Listeria were intravenously injected. Infected.
Six days after infection, the liver and spleen, which are target organs, were excised and the number of bacteria in each organ was examined. The results are shown in FIG.

As a result, the number of bacteria in each organ of the group receiving the nitric oxide production enhancer of the present invention was lower than that of the group receiving the nutritional compositions of Comparative Examples 1 and 2. .

[0025]

Example 4 Measurement of NO Production The NO production of the enhancer of the present invention was measured. That is,
Example 2 was applied to ICR mice (one group consisting of 5 mice) weighing 25 to 30 g.
After freely ingesting each composition similar to 3 and 3, for 1 week, the method of Kanazaki et al. (Biological Defense, Vol. 16, pp. 167-172 (1984))
10% thioglycolic acid medium (Eiken Chemical Co., Ltd.) according to 1.5
ml was intraperitoneally injected, and peritoneal exudate macrophages were collected on day 3. 5 × 10 ⁶ / ml of collected macrophages
LPS (lipopolysaccharide;
List Biological Laboratory) or INF-γ
(Interferon-γ; dienzyme). After culturing for 3 days, the supernatant was collected and the amount of NO produced was measured. Stein & Strejan method (Cell. Immun
ol.150: 281,1993) according to Griess reagent (Anal.Bioch
em.126: 231,1982) using, nitrite (NO ₂ ^- was measured as). The NaNO ₂ solution was used as a standard, and the measurement was performed at A ₅₅₀ . The results are shown in FIG.

As a result, NO by peritoneal macrophages
The production amount of the nitric oxide production enhancer of the present invention was higher than that of Comparative Example.

[0027]

From these results, the enhancer of the present invention is
It enhances the NO production amount of macrophages, exhibits an immunostimulatory action, and has a protective and therapeutic effect against infection.
Therefore, the present invention provides a nutritional composition useful for prevention and treatment of infectious diseases caused by postoperative invasive period, or immunodeficiency, radiation therapy, administration of anticancer agents, acquired immunodeficiency, and the like. .

[Brief description of the drawings]

FIG. 1 shows the change in the survival rate of each composition administered by intraperitoneal infection of Listeria in Example 2.

FIG. 2 shows the number of bacteria in the organs of the liver and spleen at the time of administration of each composition by intravenous administration infection of Listeria in Example 3.

FIG. 3 shows the NO production amount of peritoneal macrophages obtained from the animals to which each composition was administered in Example 4.

[Procedure amendment]

[Submission date] December 1, 1995

[Procedure amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0002

[Correction method] Change

[Correction contents]

[0002]

2. Description of the Related Art Conventionally, in determining the intake requirement of human amino acids, emphasis has been placed on balanced intake of essential amino acids, and non-essential amino acids (essential amino acids).
Was used as an index (Physiol. Rev., vo.
l. 18, pp. No. 109-136 (1938)), it has not been necessary. However, in recent years, it has been pointed out that the types and amounts of amino acids required for the living body to exhibit a physiologically optimal reaction are larger than those estimated by nitrogen equilibrium and the like. In particular, under various pathological conditions, for example, branched-chain amino acids (leucine, isoleucine, valine) in the invasive period (hereinafter, all amino acids not specified are L-type amino acids except glycine) and glutamine required amount. Increase. And
Seifter et al. Have shown that ingestion of arginine shortens the number of days for wound healing, which indicates that arginine is converted into proline, which is a constituent of collagen required for granulation, and is supplied to wound healing. It is reported that this is because it has an effect of stimulating the secretion of hormones such as insulin and glucagon.
rgery, vol. 84, pp. 224-230 (1
978)). In addition, Barbul et al. Have observed enhancement of immune function by administering 30 g of arginine to normal persons (Surgery, vol. 90, pp. 244-25.
1 (1981)), and Reynolds et al. Also reported that arginine has an effect on immunity such as promotion of differentiation of cytotoxic T lymphocytes (Surger).
y, vol. 104, pp. 142-151 (198
8)). These facts suggest that arginine is not only a constituent of somatic proteins, but also necessary for maintaining physiological functions. Therefore, the amount of arginine required to maintain the living body is not accurate according to the Rose standard, and is considered to be higher (Yasuyoshi Wakabayashi et al., Surgery and Metabolism and Nutrition, vol. 25, pp . 3 98-404).
(1991)). Therefore, if the amount of arginine synthesized in the body decreases due to diseases, etc., or if the amount of arginine supplied in nutritional management during the disease stage is less than the required amount,
There is a concern that delay in healing and recovery from disease, exacerbation of symptoms, complications, etc. may occur.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0020

[Correction method] Change

[Correction contents]

[0020]

[Table 6]

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] 0025

[Correction method] Change

[Correction contents]

[0025]

Example 4 Measurement of NO Production The NO production of the caulking agent of the present invention was measured. That is,
After the ICR strain mice (1 group 5 mice) in Examples 2 and 3 the same respective compositions in weight 25~30g libitum for one week, gold Ke Sakira methods (biodefense, Volume 1, 167- 172
Page (1984)), 1.5 ml of 10% thioglycolic acid medium (Eiken Chemical Co., Ltd.) was intraperitoneally injected, and peritoneal exudate macrophages were collected on the third day. Adjust the collected macrophages to be 5 × 10 ⁶ / ml, and add L
PS (lipopolysaccharide; List Biological Laboratory) or INF-γ (interferon-
γ: Dienzyme) was used for stimulation. After culturing for 3 days, the supernatant was collected and the amount of NO produced was measured. NO production is Ste
in &Strejan's method (Cell. Immuno
l. 150: 281, 1993).
reagent (Anal. Biochem. 12
6: 231, 1982) using the nitrite (N
It was measured as O ₂ ⁻ ). As a standard, a NaNO ₂ solution was used and measurement was performed at A ₅₅₀ . The results are shown in FIG.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display area // (A61K 31/195 31:40)

Claims (5)

[Claims] 1. A nitric oxide production enhancer for macrophages, comprising an amino acid composition containing 10 to 30% by weight of arginine as an active ingredient. 2. The enhancer according to claim 1, wherein the amino acid composition satisfies the following conditions (1) to (4). (1) The ratio (% by weight) of essential amino acids to all amino acids in the amino acid composition is as follows. L-isoleucine 4.0 to 8.0 L-leucine 7.0 to 14.0 L-lysine 5.0 to 10.0 L-methionine and L-cystine 3.0 to 6.0 L-phenylalanine and L-tyrosine 6.0 to 9.0 L-threonine 4.0 to 6.0 L-tryptophan 1.0 to 2.0 L-valine 5.0 to 10.0 L-histidine 2.0 to 4.0 (2) The ratio of L-arginine to all amino acids in the amino acid composition is 10 to 30% by weight. (3) The weight ratio of L-arginine to L-lysine in the amino acid composition is 1 to 4. (4) The ratio (% by weight) of non-essential amino acids excluding L-arginine to all amino acids in the amino acid composition is as follows. L-alanine 2.5 to 10.0 L-aspartic acid and L-asparagine 6.0 to 11.0 L-glutamic acid and L-glutamine 7.0 to 21.0 Glycine 1.0 to 8.0 L-proline 3.0 to 11.0 L-serine 2.0 to 7.0 3. The enhancer according to claim 1, wherein the amino acid composition is an amino acid mixture. 4. The enhancer according to claim 1, wherein the amino acid composition is a protein hydrolyzate based on which the amino acid composition is adjusted by adding free amino acids or peptides. 5. Use for prevention and treatment of infectious diseases.
The enhancer according to any one of to 4.