JP2007297379A - Functional activator of dendritic cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a more effective functional activator of dendritic cell and to provide a method for producing dendritic cells having an activated function. <P>SOLUTION: The functional activator of dendritic cell comprises at least one branched-chain amino acid selected from isoleucine, leucine and valine as an active ingredient. The method for producing dendritic cells having an activated function comprises culturing dendritic cells or their precursor cells in a medium containing the activator. The dendritic cells having an activated function are provided by the method. The prophylactic or therapeutic agent comprises the cells. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a dendritic cell function activator and a food effective for dendritic cell function activation. The present invention relates to the field of cancer prevention / treatment using dendritic cells with activated functions.

  Dendritic cells (DCs) activate unsensitized helper T cells and cytotoxic T cells as antigen-presenting cells, and also act on B cells, natural killer (NK) cells, and NKT cells. It plays a central role in the innate and acquired immune systems. Dendritic cells are attracting attention as the most powerful antigen-presenting cells and are thought to play an important role in biological defense. Therefore, it is considered that when the dysfunction occurs in dendritic cells, the immune system is seriously broken. For example, the number of dendritic cell progenitor cells is decreased in patients with acquired immunodeficiency syndrome (AIDS), and this may be one factor causing immunodeficiency (Non-patent Document 1).

  It has been conventionally pointed out that patients with cirrhosis have a reduced immune system function. It is known that the function of dendritic cells also decreases, and this is considered to be one of the causes that hepatitis C virus (HCV) and hepatitis B virus suppress the function of dendritic cells. (Non-patent document 2). Moreover, patients with cirrhosis often develop infections such as idiopathic bacterial peritonitis due to the reduced function of dendritic cells. In addition, it is known that hepatitis C recurs frequently in liver transplantation in patients with liver cirrhosis caused by hepatitis C virus. However, the decrease in the function of dendritic cells and the recurrence rate may be related. It is known (Non-patent Document 3). On the other hand, in patients with cirrhosis, the balance of various amino acids in the blood is lost as the disease progresses, branched-chain amino acids (isoleucine, leucine, valine: BCAA) are decreased, and aromatic amino acids (phenylalanine, tryptophan, tyrosine: AAA) are increased. It is known to do.

  Molecular biological approach reveals that free amino acids not only become physiologically active substances and cellular components, but also have physiological activities themselves and play an important role in the expression of various cellular functions It is being done. For example, it has been reported that branched chain amino acids can improve neutrophil phagocytosis and NK activity in patients with cirrhosis (Non-patent Document 4).

In addition, dendritic cells are attracting attention not only for in vivo immune responses but also for their usefulness, such as in vitro differentiation induction and use in immunotherapy (adoptive immunotherapy, etc.). Various cytokines are involved in the activation of dendritic cells. As a method for differentiating dendritic cells in vitro, a method of differentiating mononuclear cells or immature dendritic cells collected from peripheral blood using cytokines such as GM-CSF, IL-4, and TNF is known. However, these cytokines are expensive and cause side effects when applied to living bodies. Therefore, development of an effective and safe method for activating dendritic cells is desired.
Blood, Vol. 98, p906 (2001) Lancet Infect Dis., Vol.5, p296-p304 (2005) Transplant Proc., Vol.37, p3951-3956 (2005) Hepatology Research, Vol. 29, p207-p211 (2004) J.Gen.Virol., Vol.87, p1-10 (2006)

  An object of the present invention is to provide an effective dendritic cell function activator that is inexpensive and has few side effects.

  As a result of diligent research to solve the above-mentioned problems, the inventors of the present invention activate a dendritic cell function using a composition containing at least one branched chain amino acid selected from isoleucine, leucine and valine as an active ingredient. As a result, the present invention has been completed.

That is, the present invention provides the following.
(1) A dendritic cell function activator comprising, as an active ingredient, at least one branched chain amino acid selected from isoleucine, leucine and valine;
(2) The agent according to (1) above, containing isoleucine, leucine and valine as active ingredients;
(3) The agent according to (2) above, wherein the weight ratio of isoleucine, leucine and valine is 1: 1.5 to 2.5: 0.8 to 1.7;
(4) The agent according to (1) above, containing valine as an active ingredient;
(5) The agent according to any one of the above (1) to (4), which is a medicine;
(6) The agent according to (5) above, which is used for prevention / treatment of infectious diseases;
(7) The agent according to (6) above, wherein the infectious disease is idiopathic bacterial peritonitis;
(8) A package in which the dendritic cell functional activator according to any one of (1) to (7) is packaged together with a description that describes matters relating to use for functional activation of dendritic cells;
(9) It contains at least one kind of branched chain amino acid selected from isoleucine, leucine and valine, has a function activating function of dendritic cells, and is used for activating the functions of dendritic cells. Food with a label indicating that it is a product;
(10) The food according to (9) above, wherein the intake is 0.5 to 30.0 g isoleucine, 1.0 to 60.0 g leucine, and 0.5 to 30.0 g valine per day;
(11) The food according to (9) above, containing isoleucine, leucine and valine;
(12) The food according to (11) above, wherein the intake is 2 to 50 g per day in terms of total of isoleucine, leucine and valine;
(13) The food according to (9) above, containing valine;
(14) The food according to (13), wherein the intake is 0.5 to 30.0 g of valine per day;
(15) The food according to any one of (9) to (14) above, wherein the food is a health functional food or a dietary supplement;
(16) The food according to (15) above, wherein the health functional food is a food for specified health use or a food with a nutritional function;
(17) A package in which a food containing isoleucine, leucine and valine is packaged together with a description describing matters relating to use for functional activation of dendritic cells;
(18) A package in which a food containing valine is packaged together with a description describing matters relating to use for functional activation of dendritic cells;
(19) A method for producing a dendritic cell having an activated function, wherein the dendritic cell or its precursor cell is cultured in a medium to which the agent according to any one of (1) to (4) above is added. A method comprising:
(20) A dendritic cell having an activated function produced by the method according to (19) above;
(21) A cancer preventive / therapeutic agent comprising the cell according to (20) above;
(22) A method for preventing and / or treating cancer, comprising the following steps:
(A) a step of culturing a dendritic cell or a progenitor cell thereof in a medium to which the agent according to any one of (1) to (4) is added;
(B) allowing the cells cultured in the step (a) to recognize a cancer antigen, and (c) administering the dendritic cells recognized in the antigen in the step (b) to the subject,
Including methods.

The dendritic cell function activator provided by the present invention and containing at least one branched chain amino acid selected from isoleucine, leucine and valine as an active ingredient is used to activate the function of dendritic cells. The activator of the present invention is extremely advantageous as a pharmaceutical because it has an amino acid as an active ingredient and is highly safe and has few side effects.
In addition, since the three types of branched chain amino acids of isoleucine, leucine and valine, which are active ingredients in the activator of the present invention, are substances with established safety, the activator of the present invention has high safety, Therefore, it can be used for food as well as for pharmaceutical use.
Furthermore, the dendritic cells produced in the medium supplemented with the activator of the present invention are very useful for use in immunotherapy and the like because their functions are activated.

  The present invention provides a dendritic cell function activator (hereinafter, the activator of the present invention).

  In the present specification, “function of dendritic cells” refers to the function of dendritic cells as antigen-presenting cells, the ability to produce cytokines, and specifically to the ability to activate T lymphocytes. “Functional activation of dendritic cells” refers to the proliferating action of T lymphocytes by antigen-presenting dendritic cells. "Functional activation of dendritic cells" measures increase in lymphocyte proliferative capacity by MLR (Mixed Leukocyte Reaction) assay or changes in phenotype of dendritic cells by FACS, as described in Examples below. This can be confirmed by a known method (Non-Patent Document 5).

The active ingredient (branched chain amino acid) of the present invention, isoleucine, leucine and valine, can be used in any of L-form, D-form and DL-form, respectively, preferably L-form and DL-form. -Form, more preferably L-form.
Further, isoleucine, leucine and valine can be used not only in free form but also in salt form. Examples of the salt form include acid addition salts and salts with bases, and it is preferable to select pharmaceutically acceptable salts of isoleucine, leucine and valine.
Examples of acids that are added to isoleucine, leucine, and valine to form pharmaceutically acceptable salts include inorganic acids such as hydrogen chloride, hydrogen bromide, sulfuric acid, and phosphoric acid; acetic acid, lactic acid, citric acid, tartaric acid, Organic acids such as maleic acid, fumaric acid, monomethyl sulfuric acid and the like can be mentioned.
Examples of bases that form pharmaceutically acceptable salts of isoleucine, leucine and valine include, for example, metal hydroxides or carbonates such as sodium, potassium and calcium, or inorganic bases such as ammonia; ethylenediamine, propylenediamine And organic bases such as ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine.

  The activator of the present invention contains at least one branched chain amino acid selected from isoleucine, leucine and valine as an active ingredient. The activator of the present invention preferably contains valine as an active ingredient. When the activator of the present invention contains isoleucine, leucine, and valine, the mixing ratio of the three amino acids is usually in the ratio of 1: 1.5 to 2.5: 0.8 to 3, respectively. 0.0, particularly preferably in the range of 1: 1.9 to 2.2: 1.1 to 1.3. Outside this range, it is difficult to obtain an effective action effect.

  The activator of the present invention is useful as a medicine and the like, and examples of the administration target include mammals (eg, human, mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, etc.). . In addition, when adapting to mammals other than a human, the intake of the activator of this invention should just be adjusted suitably according to the body weight or magnitude | size of an animal.

  Here, about the activator of this invention, the administration form in the case of using as a pharmaceutical is not specifically limited, However, It can pass through common administration routes, such as oral administration, rectal administration, injection, and administration by infusion. Oral dosage forms include granules, fine granules, powders, coated tablets, tablets, suppositories, powders, (micro) capsules, chewables, syrups, juices, liquids, suspensions, emulsions, etc. In addition, general dosage forms of pharmaceutical preparations such as direct intravenous injection, infusion administration, and preparations that prolong the release of active substances can be adopted as injections.

  These medicaments are prepared by formulating them by a conventional method. Various pharmaceutical substances that are pharmacologically acceptable can be blended as required in the preparation. The substance for the preparation can be appropriately selected depending on the dosage form of the preparation. For example, the excipient, the diluent, the additive, the disintegrant, the binder, the coating agent, the lubricant, the lubricant, the lubricant, and the flavoring agent. , Sweeteners, solubilizers and the like. Furthermore, specific examples of the pharmaceutical substance include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, cellulose and derivatives thereof, animal and vegetable oils, polyethylene glycol, and solvents, Examples include sterilized water and mono- or polyhydric alcohols such as glycerol.

  When the activator of the present invention is used as a pharmaceutical, the dose varies depending on the age, weight or disease state, dosage form, administration method, etc. of the subject patient. 5 to 30.0 g, leucine 1.0 to 60.0 g, and valine 0.5 to 30.0 g as a guide. In the case of general adults, preferably, as a daily dose, isoleucine 2.0-10.0 g, leucine 3.0-20.0 g, valine 2.0-10.0 g, more preferably isoleucine 2.5- 3.5 g, leucine 5.0 to 7.0 g, and valine 3.0 to 4.0 g, and the total amount of amino acids is preferably about 2.0 g to 50.0 g per day. The daily dose can be administered at once or in several divided doses. It does not matter before meals, after meals, and between meals. The administration period is not particularly limited.

When calculating the dose (intake) of the branched chain amino acid that is the active ingredient of the present invention, the value is the amount of the active ingredient of the drug used for the purpose of treatment or prevention of the disease targeted by the present invention. As for the branched chain amino acids to be ingested or administered for other purposes, for example, from the necessity of normal eating habits, or for the purpose of treating other diseases. Need not be included in the calculation.
For example, it is not necessary to subtract the amount of branched chain amino acids per day taken from the normal diet from the daily dose of the active ingredient in the present invention.

  Isoleucine, leucine and valine, which are the active ingredients of the present invention, may be contained in the preparation alone or in any combination, or all may be contained in one kind of preparation. When separately formulated and administered, their administration route and dosage form may be the same or different, and the timing of administration of each may be the same or different. It is determined as appropriate depending on the type and effect of the drug used in combination. That is, the pharmaceutical of the present invention may be a preparation containing a plurality of branched chain amino acids at the same time, or may be a combined preparation in which each is separately formulated and used together. All these forms are included. In particular, an embodiment containing all branched chain amino acids in the same preparation is preferable because it can be easily administered.

  In the present invention, the “weight ratio” indicates the weight ratio of each component in the preparation. For example, when each active ingredient of isoleucine, leucine and valine is included in one preparation, it is the ratio of individual contents, and each active ingredient is included in multiple preparations alone or in any combination The ratio of the weight of each active ingredient included in each formulation.

  In the present invention, the actual dose ratio is a ratio of a single dose or a daily dose of each active ingredient per administration subject (for example, a patient). For example, when each active ingredient of isoleucine, leucine and valine is included in one preparation and is administered to an administration subject, the weight ratio corresponds to the dose ratio. When each active ingredient is administered alone or in any combination in a plurality of preparations, the ratio of the total amount of each active ingredient in each preparation administered once or daily corresponds to the weight ratio.

  The medicament of the present invention may be blended with other drugs such as anticancer agents, antiviral agents, antibiotics, liver disease agents, immunosuppressive agents and the like.

Isoleucine, leucine, and valine have already been widely used in the pharmaceutical and food fields, and safety has been established. For example, the acute toxicity (LD ₅₀ ) of a formulation containing these amino acids in a ratio of 1: 2: 1.2 is 10 g / kg or more upon oral administration to mice.

  In the present invention, at least one kind of branched chain amino acid selected from isoleucine, leucine and valine as an active ingredient, the dendritic cell functional activator can be used for functional activation of dendritic cells. Or a commercial package containing a statement stating that it should be used.

The activator of the present invention activates the function of dendritic cells. Since activation of dendritic cell function is closely related to activation of immune function, the activator of the present invention is also useful for prevention and / or treatment of immune-related diseases. Examples of immune-related diseases include infectious diseases (bacteremia, pneumonia, urinary tract infection, idiopathic bacterial peritonitis, idiopathic bacterial empyema, bacterial meningitis, sepsis, tuberculous peritonitis, and the like). In patients with liver cirrhosis, various infections and idiopathic bacterial peritonitis occur frequently due to the reduced function of dendritic cells. Therefore, since the activator of the present invention activates the function of dendritic cells, it is useful for the prevention and / or treatment of various infections and idiopathic bacterial peritonitis in cirrhotic patients.
In addition, when liver transplantation is performed in patients with type C cirrhosis, it is known that hepatitis virus recurs frequently in the transplanted liver, but hepatitis virus recurrence can be achieved by activating the function of dendritic cells. It is useful for prevention.

  The present invention also includes a food containing at least one branched chain amino acid selected from isoleucine, leucine, and valine and having a label used for activating the function of dendritic cells (hereinafter referred to as the food of the present invention). )I will provide a.

As the branched chain amino acids contained in the food of the present invention, isoleucine, leucine and valine can be used in any of L-form, D-form and DL-form, respectively, preferably L-form and DL-form. -Form, more preferably L-form.
Further, isoleucine, leucine and valine can be used not only in free form but also in salt form. Examples of the salt form include acid addition salts and salts with bases, and it is preferable to select pharmaceutically acceptable salts of isoleucine, leucine and valine.
Examples of acids that are added to isoleucine, leucine, and valine to form pharmaceutically acceptable salts include inorganic acids such as hydrogen chloride, hydrogen bromide, sulfuric acid, and phosphoric acid; acetic acid, lactic acid, citric acid, tartaric acid, Organic acids such as maleic acid, fumaric acid, monomethyl sulfuric acid and the like can be mentioned.
Examples of bases that form pharmaceutically acceptable salts of isoleucine, leucine and valine include, for example, metal hydroxides or carbonates such as sodium, potassium and calcium, or inorganic bases such as ammonia; ethylenediamine, propylenediamine And organic bases such as ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine.

  The food of the present invention contains at least one branched chain amino acid selected from isoleucine, leucine and valine. In particular, the food of the present invention preferably contains valine. When the food of the present invention contains isoleucine, leucine and valine, the blending ratio of these three amino acids is usually 1: 1.5 to 2.5: 0.8 to 3.0 by weight ratio. And particularly preferably in the range of 1: 1.9 to 2.2: 1.1 to 1.3. Outside this range, it is difficult to obtain an effective action effect.

The “food” of the present invention may be any general food form containing at least one branched chain amino acid selected from isoleucine, leucine and valine. For example, drinks such as soft drinks and powdered drinks can be prepared by adding an appropriate flavor. Specifically, for example, juice, milk, confectionery, jelly, yogurt, rice cake, etc. can be mixed with at least one branched chain amino acid selected from isoleucine, leucine and valine to eat and drink.
It is also possible to provide such foods as health functional foods or dietary supplements. This health functional food includes food for specified health use and food with nutritional function. The food for specific health is a food that can indicate that a specific health purpose can be expected, for example, functional activation of dendritic cells. In addition, functional nutritional food is a food that can indicate the function of the nutritional component when the amount of nutritional component included in the daily intake standard amount conforms to the national and national standards for upper and lower limits. is there. Dietary supplements include so-called nutritional supplements or health supplements. In the present invention, the food for specified health use is a food with an indication that it is used for applications such as functional activation of dendritic cells, and further a document (so-called so-called document) stating that it is used for such use. Food and the like that include a package).
Furthermore, the food of the present invention can be used as a concentrated liquid food or a food supplement. When used as a food supplement, it can be prepared in the form of tablets, capsules, powders, granules, suspensions, chewables, syrups and the like. The food supplement in the present invention refers to those taken for the purpose of supplementing nutrition in addition to those taken as food, and also includes nutritional supplements and supplements.

  In addition, “rich liquid food” is adjusted to a concentration of about 1 kcal / ml, and the qualitative composition of each nutrient is sufficiently considered so that no significant excess or deficiency of nutrients is caused even by long-term single intake. It is a comprehensive nutritional food (liquid food) designed based on the nutritional requirements of food.

  When ingested as food, the intake varies depending on the symptom, age, weight, dosage form, intake method, etc. of the subject, but adults (with a body weight of 60 kg) per day, 0.5 to 30.0 g isoleucine, 1. The standard is 0 to 60.0 g and valine 0.5 to 30.0 g. In the case of general adults, preferably, as a daily dose, isoleucine 2.0-10.0 g, leucine 3.0-20.0 g, valine 2.0-10.0 g, more preferably isoleucine 2.5- 3.5 g, leucine 5.0 to 7.0 g, and valine 3.0 to 4.0 g, and the total amount of amino acids is preferably about 2.0 g to 50.0 g per day. In the food of the present invention, the amount per day can be taken at once or divided into several times. The intake period is not particularly limited.

Isoleucine, leucine, and valine have already been widely used in the pharmaceutical and food fields, and safety has been established. For example, the acute toxicity (LD ₅₀ ) of a formulation containing these amino acids in a ratio of 1: 2: 1.2 is 10 g / kg or more upon oral administration to mice.

  In the present invention, it is described that a food containing at least one kind of branched chain amino acid selected from isoleucine, leucine and valine can or should be used for functional activation of dendritic cells. Commercial packages are also included, including descriptions.

The food of the present invention activates the function of dendritic cells. Since the activation of the function of dendritic cells is closely related to the activation of the immune function, the food of the present invention is also useful for the prevention of immune-related diseases. Examples of immune-related diseases include infectious diseases (bacteremia, pneumonia, urinary tract infection, idiopathic bacterial peritonitis, idiopathic bacterial empyema, bacterial meningitis, sepsis, tuberculous peritonitis, and the like). In patients with liver cirrhosis, various infections and idiopathic bacterial peritonitis occur frequently due to the reduced function of dendritic cells. Therefore, since the food of the present invention activates the function of dendritic cells, it is useful for preventing various infectious diseases and idiopathic bacterial peritonitis in cirrhosis patients.
In addition, when liver transplantation is performed in patients with type C cirrhosis, it is known that hepatitis virus recurs frequently in the transplanted liver, but hepatitis virus recurrence can be achieved by activating the function of dendritic cells. It is useful for prevention.

  The present invention also provides a method for producing a dendritic cell whose function is activated using the activator of the present invention (hereinafter, the production method of the present invention).

In the production method of the present invention, dendritic cells or progenitor cells separated from a living body are cultured in a medium to which the activator of the present invention is added. Examples of the living body include warm-blooded animals, preferably mammals. Examples of mammals include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, monkeys, humans, etc., preferably mice, rats, monkeys, humans, etc. Is mentioned. Dendritic cells or their precursor cells are preferably isolated from peripheral blood. Examples of progenitor cells include CD14 ⁺ mononuclear cells, CD34 ⁺ hematopoietic stem cells, and the like, and preferably CD14 ⁺ mononuclear cells and the like, and the progenitor cells are prepared from living bodies by a method known per se.

  The medium in the production method of the present invention can be prepared by using a medium used for culturing animal cells as a basal medium and adding the activator of the present invention. Examples of the basal medium include Eagle MEM medium, αMEM medium, DMEM medium, ham medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof. The medium may be, for example, serum (for example, FCS), serum replacement (for example, knockout Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), Glutamax (Gibco)), fatty acid or lipid, amino acid, Vitamins, growth factors, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts and the like can be included. Preferably, the medium is DMEM medium or RPMI 1640 medium.

  The content of the activator of the present invention in the culture medium is adjusted to be 1 mg / l to 2 g / l of isoleucine, 1 mg / l to 2 g / l of leucine, and 1 mg / l to 2 g / l of valine. Preferred are isoleucine 20 mg / l to 500 mg / l, leucine 20 mg / l to 500 mg / l, and valine 20 mg / l to 500 mg / l.

Other culture conditions such as culture temperature, CO ₂ concentration, and culture time in the production method of the present invention can be set as appropriate. The culture temperature is not particularly limited and is, for example, about 30 to 40 ° C, preferably about 37 ° C. The CO ₂ concentration is, for example, about 1 to 10%, preferably about 5%. The culture time is, for example, about 1 day to 14 days, preferably about 6 days.

  Since the dendritic cells produced by the production method of the present invention have activated functions such as antigen-presenting ability, they can be used for immunotherapy, prevention and / or treatment of immune-related diseases. Immunotherapy includes cancer therapy, and immune-related diseases include infection (bacteremia, pneumonia, urinary tract infection, idiopathic bacterial peritonitis, idiopathic bacterial empyema, bacterial meningitis, sepsis And tuberculous peritonitis).

  Therefore, the present invention provides a preventive / therapeutic agent for cancer comprising dendritic cells produced by the production method of the present invention as an active ingredient. The cancer preventive / therapeutic agent of the present invention can be administered to a patient, etc. to prevent cancers such as melanoma, leukemia, thyroid cancer, liver cancer, pancreatic cancer, colon cancer, lung cancer, gastric cancer, gallbladder / bile duct cancer, breast cancer, uterine cancer, etc. It can be usefully used for prevention and treatment.

  For example, dendritic cells or progenitor cells thereof are collected from cancer patient blood, and dendritic cells derived from the patient are produced by the method of the present invention. Then, by culturing the dendritic cells together with the cancer-specific antigen peptide or the destroyed cancer cells, the ability to induce immunity against cancer is enhanced and then administered to the patient, thereby preventing or treating cancer. . As a cancer antigen peptide, a lysate of a cancer patient's own cancer tissue or a known antigen peptide can be synthesized and used.

  The prophylactic / therapeutic agent for cancer of the present invention is usually prepared by mixing an effective amount of dendritic cells with a pharmaceutically acceptable carrier according to a conventional means, and is usually non-injectable such as an injection, suspension, infusion, etc. Manufactured as an oral formulation. Examples of carriers that can be included in the parenteral preparation include aqueous solutions for injection such as physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.). A liquid can be mentioned. Examples of the preventive / therapeutic agent for cancer of the present invention include a buffer (for example, phosphate buffer, sodium acetate buffer, etc.), a soothing agent (for example, benzalkonium chloride, procaine, etc.), a stabilizer (for example). For example, human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants and the like may be blended.

  The preparation thus obtained can be administered, for example, to the above-mentioned mammals such as humans by instillation, local administration or the like.

The dose of the dendritic cell of the present invention varies depending on the administration subject, target organ, symptom, administration method and the like. Usually, in an adult patient (with a body weight of 60 kg), for example, in the case of parenteral administration, It is convenient to administer about 1 × 10 ⁶ cells to 1 × 10 ⁹ cells per cell. In the case of other animals, an amount converted per 60 kg body weight can be administered.

  The contents of all publications, including patents and patent application specifications cited in this specification, are hereby incorporated by reference herein to the same extent as if all were explicitly stated. Is.

  The present invention will be described more specifically by the following examples. However, the examples are merely illustrative of the present invention and do not limit the scope of the present invention.

Test example 1
CD14 ⁺ monocytes were collected with magnetic beads from the peripheral blood of healthy subjects and patients with chronic hepatitis C / cirrhosis. In this study, since accurate evaluation of the amino acid concentration in the medium is necessary, a unique medium (amino acid-free D-MEM medium + 20 kinds of amino acids + various additives) was prepared, and a medium containing all 20 kinds of amino acids. Was defined as Full medium (Complete Culture Medium: CCM) and used as control. In addition, the amino acid composition of the Full medium was measured using high performance liquid chromatography, and it was confirmed that there was no difference between the theoretical value and the actual measurement value.

CD14 ⁺ monocytes were cultured for 5 days in a Full medium supplemented with GM-CSF, IL-4 and TNF-α, and LPS was added on the 5th day, followed by additional culture for 24 hours. Differentiation into dendritic cells was confirmed by FACS using CD14 ⁻ , CD40 ⁺ , CD80 ⁺ , CD83 ⁺ , CD86 ⁺ , HLADR ⁺ , and a system capable of differentiation into dendritic cells was established.

Media in which one type of amino acid has been removed from Full media (ex. ΔGly, ΔAla, ΔSer, ΔThr, ΔCys, ΔMet, ΔGln, ΔAsn, ΔGlu, ΔAsp, ΔVal, ΔLeu, ΔIle, ΔPhe, ΔTyr, ΔTrp, ΔLys, Arg, , ΔHis, ΔPro total 20 types), medium from which several types of amino acids have been removed (ex. ΔBCAA (ΔVal, ΔLeu, ΔIle), ΔAAA (ΔPhe, ΔTyr, ΔTrp), ΔaAA (ΔGlu, ΔAsp), ΔbAA (ΔLys, ΔArg, ΔHis), ΔeAA (ΔThr, ΔMet, ΔVal, ΔLeu, ΔIle, ΔPhe, ΔTrp, ΔLys, ΔHis), ΔneAA (ΔGly, ΔAla, ΔSer, ΔCys, ΔGln, ΔAsn, ΔGlu, ΔAsp, Δglu, ΔAsp, Δg , Δs A (ΔCys, ΔMet), ΔNH2AA (ΔGln, ΔAsn) eight kinds), all amino acids were removed medium (Zero medium) were cultured CD14 ⁺ monocytes at a total 30 kinds of media Full medium (CCM) . In addition, Table 1 shows the amino acid content in each medium of Full medium, ΔBCAA medium, ΔVal medium, ΔLeu medium, and ΔIle medium. After irradiation on the 6th day, CD4 ⁺ T-cell was added, and the cells were further cocultured for 4 days under Full medium (CCM). Focusing on BCAA, branched chain amino acid-removed medium (ΔBCAA), valine-removed medium (ΔVal), and leucine-removed medium (ΔLeu) have significantly reduced lymphocyte proliferation ability compared to Full medium (CCM). (FIG. 1). That is, co-culture with dendritic cells cultured and differentiated in these removal media showed that the activation of lymphocytes was weak, and CD4 ⁺ lymphocyte stimulating ability of monocyte-derived dendritic cells (antigen) It is conceivable that the presentation ability has been reduced.

On day 6 of culture, the phenotype of the dendritic cells (CD14, CD40, CD80, CD83, CD86) was measured using FACS. ΔBCAA significantly decreased CD83 and CD86 (FIG. 3). Furthermore, a decrease tendency of CD83 and CD86 was observed with ΔVal, but not with ΔLeu and ΔIle. 2 Color staining revealed that the ratio of mature dendritic cells was decreased in ΔBCAA and ΔVal.
When the dendritic cell recovery rate on the sixth day of culture was measured (trypan blue, FACS), ΔBCAA, ΔVal, and ΔLeu all decreased by about 10%.

With respect to Valine and Leucine, CD14 ^{+ of} dendritic cells of healthy subjects and C-type cirrhosis patients in the same manner with concentration gradients of removal (Δ), × 1 (Full), × 2, × 3, × 5, and × 10. Monocytes were cultured (FIG. 2). With respect to Valine, the proliferation ability of lymphocytes increased in a dose-dependent manner up to 3 times the concentration (× 3) in both healthy subjects and patients, and reached the maximum at 3 times the concentration (FIG. 2 (B)). .

Test example 2
Using CD14 ⁺ monocytes collected with magnetic beads from the peripheral blood of healthy subjects and patients with chronic hepatitis C / cirrhosis, measurement of lymphocyte proliferation ability was repeated in the same manner as in Test Example 1. Focusing on BCAA, the branched chain amino acid-removed medium (ΔBCAA) and the valine-removed medium (ΔVal) significantly decreased the lymphocyte proliferation ability compared to the Full medium (CCM) (FIG. 4A). That is, co-culture with dendritic cells cultured and differentiated in these removal media showed that the activation of lymphocytes was weak, and CD4 ⁺ lymphocyte stimulating ability of monocyte-derived dendritic cells (antigen) It is conceivable that the presentation ability has been reduced. In this test example, the leucine removal medium (ΔLeu) system showed a tendency to decrease the lymphocyte proliferative ability, but no significant difference was obtained.

In addition, when a similar experiment was conducted using CD14 ⁺ monocytes of healthy individuals, and the lymphocyte proliferative ability was measured under the conditions where Val and Leu were added to ΔVal medium and ΔLeu medium at various concentrations, Val was dependent on the concentration. It was revealed that lymphocytes were activated by acting on dendritic cells (FIG. 4B). Furthermore, IL-12 and IL-10 cytokine concentrations in the dendritic cell culture medium on day 5 under the same culture conditions were measured using a specific ELISA kit (Bender MedBiosystems), depending on the Val concentration. Although IL-10 production was not affected, it was revealed that IL-12 production increased (FIG. 4C). IL-12 is a cytokine produced mainly from macrophages / dendritic cells, and enhances the differentiation of naive T cells into Th1 cells and the cytotoxic activity of NK cells and CD8 ⁺ T cells. Therefore, enhancement of IL-12 production means functional differentiation of dendritic cells. Moreover, since IL-12 acts on antigen-sensitized T cells and induces infiltration into inflamed areas (including cancer masses), the production enhancement is significant from the viewpoint of enhancing the antitumor effect.

With respect to Valine, Val was added to the ΔVal medium with a concentration gradient, and CD14 ⁺ monocytes of healthy subjects and type C cirrhosis patients were cultured in the same manner (FIG. 5). With respect to Valine, the proliferation ability of lymphocytes increased in a dose-dependent manner in both healthy subjects and patients (FIG. 5 (A)). Furthermore, at this time, cytokines in the dendritic cell culture medium were measured, and as a result of healthy subjects (FIG. 4C), IL-12 selective production enhancement was observed also in patient dendritic cells ( FIG. 5 (B)).

  From the above results, it is shown that branched chain amino acids, especially Val, are useful for activating the function of dendritic cells since lymphocyte activation is promoted by quantification using allo-MLR as an index by dendritic cells. It was.

It is a figure which shows the lymphocyte proliferation ability in the culture medium which changed the containing amino acid. (Full: Full medium, Zero: Amino acid-free medium, ΔAAA: Phenylalanine, tryptophan and tyrosine removed medium, ΔBCAA: Isoleucine, leucine and valine removed medium, ΔIle: Isoleucine removed medium, ΔLeu: Leucine removed medium, ΔPhe: Phenylalanine removed medium , ΔTrp: tryptophan removal medium, ΔTyr: tyrosine removal medium, ΔVal: valine removal medium) It is a figure which shows the lymphocyte proliferation ability in the leucine (A) and valine (B) containing culture medium of each density | concentration. It is a figure which shows the phenotype of the CD14 ⁺ monocyte of culture | cultivation day 0, and the dendritic cell of the culture | cultivation day 6. It is a figure which shows the lymphocyte proliferation ability of the healthy human dendritic cell in the culture medium which changed the content amino acid. (CCM: CCM medium, Zero: amino acid-free medium, ΔBCAA: isoleucine, leucine and valine removed medium, ΔVal: valine removed medium, ΔLeu: leucine removed medium, ΔIle: isoleucine removed medium) It is a figure which shows the lymphocyte proliferation ability of the healthy human dendritic cell in the culture medium which changed the content amino acid concentration. (○: Valine, ◆: Leucine) It is a figure which shows the cytokine production enhancement of the healthy human dendritic cell by valine. It is a figure which shows the healthy person ((circle)) and C-type cirrhosis patient ((circle)) lymphocyte proliferative ability in the culture medium which changed the containing valine density | concentration. It is a figure which shows the cytokine production enhancement of the dendritic cell of a C-type cirrhosis patient by valine.

Claims (21)

  A dendritic cell function activator comprising at least one branched chain amino acid selected from isoleucine, leucine and valine as an active ingredient.   The agent according to claim 1, comprising isoleucine, leucine and valine as active ingredients.   The agent according to claim 2, wherein the weight ratio of isoleucine, leucine and valine is 1: 1.5 to 2.5: 0.8 to 1.7.   The agent according to claim 1, comprising valine as an active ingredient.   The agent in any one of Claims 1-4 which is a pharmaceutical.   The agent according to claim 5, which is used for prevention / treatment of infectious diseases.   The agent according to claim 6, wherein the infection is idiopathic bacterial peritonitis.   The package which packaged the function activation agent of the dendritic cell in any one of Claims 1-7 with the description which described the matter regarding the use for the function activation of a dendritic cell.   A food containing at least one kind of branched chain amino acid selected from isoleucine, leucine and valine, which is used for activating the function of dendritic cells.   The food according to claim 9, wherein the intake is 0.5 to 30.0 g of isoleucine, 1.0 to 60.0 g of leucine, and 0.5 to 30.0 g of valine per day.   The food according to claim 9, comprising isoleucine, leucine and valine.   The food according to claim 11, wherein the intake is 2 to 50 g per day in terms of total isoleucine, leucine and valine.   The food according to claim 9, comprising valine.   The food according to claim 13, wherein the intake is 0.5 to 30.0 g of valine per day.   The food according to any one of claims 9 to 14, wherein the food is a health functional food or a dietary supplement.   The food according to claim 15, wherein the health functional food is a food for specified health use or a nutritional functional food.   A package in which a food containing isoleucine, leucine and valine is packaged together with a description describing matters relating to the use for functional activation of dendritic cells.   The package which packaged the foodstuff containing a valine with the description which described the matter regarding the use for the functional activation of a dendritic cell.   A method for producing a dendritic cell having an activated function, comprising culturing a dendritic cell or a precursor cell thereof in a medium to which the agent according to any one of claims 1 to 4 is added.   A dendritic cell having an activated function produced by the method according to claim 19.   A preventive / therapeutic agent for cancer comprising the cell according to claim 20.