WO2006006272A1

WO2006006272A1 - Dendritic cell activator

Info

Publication number: WO2006006272A1
Application number: PCT/JP2005/004067
Authority: WO
Inventors: Yasunori Matsui; Yoshihiro Yamaguchi; Hiroshi Yukawa; Makoto Tamesada
Original assignee: Kobayashi Pharmaceutical Co., Ltd.
Priority date: 2004-09-30
Filing date: 2005-03-09
Publication date: 2006-01-19

Abstract

[PROBLEMS] To provide a method of safely and efficiently activating dendritic cells and provide a dendritic cell activator capable of presenting means for the method. [MEANS FOR SOLVING PROBLEMS] There is provided a dendritic cell activator comprising an extract from Cordyceps sinensis. Further, there are provided, comprising the dendritic cell activator, a pharmaceutical composition, food composition, food, beverage, etc. Still further, there is provided a method of inducing mature dendritic cells, comprising treating immature dendritic cells with the dendritic cell activator. Yet still further, there are provided maturedendritic cells obtained by the above method and a pharmaceutical composition comprising the mature dendritic cells.

Description

Specification

Dendritic cell activator

Technical field

[0001] The present application relates to a rod-shaped cell activator comprising a Cordyceps extract or a fraction thereof, particularly a rod-shaped cell maturation promoter. Furthermore, the present invention relates to an oral intake composition, pharmaceutical composition, food composition and the like containing the rod-shaped cell activator.

Background art

[0002] Spider cells are a type of immune cell having a morphological feature of having dendritic processes, and are distributed in various tissues (for example, skin and mucous membranes) to serve as immune surveillance cells. Yes. In particular, rod cells are cells that have the function of transporting antigen that has entered the body to lymphoid tissues and stimulating T cells to induce an immune response, and play a central role in the immune response together with lymphocytes.

[0003] Among the rod cells distributed in various in vivo tissues, the rod cells present in the peripheral tissue are called immature rod cells because of their low ability to activate T cells. In contrast, rod cells present in lymphoid organs are called mature rod cells because they have a strong ability to activate T cells. Such functional differences are due to increased expression of cell adhesion and costimulatory molecules (eg, CD40, CD54, CD58, CD80 and CD86) in addition to MHC (major histocompatibility complex) molecules and It is caused by changes in the production capacity of cytokines such as chemokines. In addition, as the maturation progresses, the rod-shaped cells also have a structure with strong adhesion and high motility due to reorganization of the cytoskeleton, and begin to move to the lymph nodes. In the T region of the regional lymphoid organ, the rod-like cells promote the activity of T cells by producing chemokines (Non-patent document 1: Cell engineering, No. 19, No. 9, 2000, No. 1). 1311-1317).

[0004] In vivo, maturation of rod-shaped cells is caused by TNF a produced by LPS (lipopolysaccharide), DNA, double-stranded RNA, or tissue cell force derived from induction of inflammatory response from infected bacteria or viruses. (Tumor necrosis factor a) and IL-1 (interleukin 1), as well as CD40 ligand (CD40L) expressed in active platelets, basophils, mast cells, etc. Reference 2: Banchereau J. et al., Nature, 392, 199 8 years, pp. 245-252).

[0005] In addition, in an in vitro experiment, an attempt has been made to search for a substance having a rod-like cell activation action. For example, a preparation obtained by treating Krestin (hereinafter also referred to as “rPSKj”) derived from the extract of Mycelia agaricus mycelium and an attenuated natural mutant of group A hemolytic streptococci (Su strain) with belin. Has the effect of promoting the maturation of rod cells, and that lentinan, which is commercially available as an anticancer drug containing j8-glucan derived from shiitake mushroom as an active ingredient, has no effect on rod cells (Non-Patent Document 3: Biotherapy, 15th, No. 3, 2001, pp. 385-388; Non-Patent Document 4: Cancer Immunology and Immunotherapy, 52nd, 2003, 207-214 Page;). It has also been reported that the maturation of rod-shaped cells is promoted by the cell wall skeleton of certain gram-positive bacteria (Patent Document 1: International Patent Publication 01Z048154 pamphlet). Further, it has been reported that a specific low molecular weight compound obtained by chemical synthesis has a rod-like cell activating action (Patent Document 2: Japanese Patent Application Laid-Open No. 2004-210768).

[0006] Cordyceps is a collective term for mushrooms arising from insects and the like, and is positioned as a genus of the Ascomycota fungus' Aperidomycetes · Ergotaceae. Among them, Cordyceps s inensis (Berkely) Saccardo is a mushroom that parasitizes lepidopterous bat larvae and is distributed in the alpine belts of China, Tibet, Nepal, the Himalayas, and 3000-4000 m above sea level. In China, it has been handed down as a traditional Chinese medicine for immortality and tonic. Recent studies have reported various physiological activities of the components contained in Cordyceps sinensis (Non-patent Document 7: Biol. Pharm. Bull. No.22, No.9, 1999, No.966). 970; Non-Patent Document 8: Biol. Pharm. Bull. 16th, 12th, 1993, 1291-129 3; Non-patent 9: Phytochemistry, 51st, 1999, 891-898 page). However, there has been no report on Cordyceps sinensis extract, particularly Cordycesbus sinensis extract, which has any effect on rod-shaped cells.

[0007] In addition, for the purpose of clinically applying the powerful antigen-presenting ability of rod cells, research on anti-cancer therapy and anti-infection therapy using rod cells has been advanced! (Non-Patent Document 5: Nikkei Science, February 2003, pp. 84-91; Non-Patent Document 6: Contemporary Medicine, August 2003, pp. 86-92). In such therapy, by some means It is necessary to activate the rod-shaped cells, and in particular, a safe and efficient rod-shaped cell activation agent is required.

Patent Document 1: International Patent Publication 01Z048154 Pamphlet

Patent Document 2: JP 2004-210768 A

Non-Patent Document 1: Cell Engineering, 19th, 9th, 2000, 1311-1317

Non-Patent Document 2: Banchereau J et al., Nature, No. 392, 1998, pp. 245-252 Non-Patent Document 3: Biotherapy, No. 15, No. 3, 2001, pp. 385-388

Non-patent document 4: Cancer Immunology and Immunotherapy, 52nd article, 2003

Pp. 207-214

Non-Patent Document 5: Nikkei Science, February 2003, pp. 84-91

Non-Patent Document 6: Contemporary Medicine, August 2003, pp. 86-92

Non-Patent Document 7: Biol. Pharm. Bull. No. 22, No. 9, 1999, 966-970 Non-Patent Document 8: Biol. Pharm. Bull. No. 16, No. 12, 1993, No. 1291—pages 1293 Non-Patent Document 9: Phytochemistry, 51st, 1999, 891-898

Disclosure of the invention

Means for solving the problem

[0008] The present inventor has conducted extensive research to solve the above problems, and as a result, Cordyceps extract.

In particular, the present invention was completed by discovering that an extract of Cordycesbus sinensis has a rod-like cell activating action.

[0009] An object of the present invention is to provide an extract derived from Cordyceps sinensis having a rod-like cell activation action, and a rod-like cell activator, immunostimulant, composition for oral intake, pharmaceutical composition containing the extract, It is to provide food compositions, foods and beverages.

[0010] That is, according to one aspect of the present invention, a rod-shaped cell activator comprising an extract of Cordyceps sinensis is provided. Here, the term “activation of rod cells” means that some function of rod cells is enhanced, or the amount of molecules such as proteins expressed in rod cells is amplified. The meaning of the term includes, for example, promoting maturation of rod cells, enhancing antigen uptake ability, enhancing antigen presentation ability, and enhancing T lymphocyte stimulation ability. As a phenomenon observed in rod cells with the activation, , Adhesion molecule Z co-stimulatory molecules (eg, CD40, CD54, CD58, CD80, CD86, etc.) increased expression, and site force-in (eg, interleukin 12, interferon gamma and interleukin 1) And increase the expression level of MHC molecules.

[0011] According to another aspect of the present invention, there is provided a pharmaceutical composition comprising the above rod-shaped cell activator. The pharmaceutical composition is not particularly limited, and can be used, for example, for immunostimulation, treatment or prevention of cancer or malignant tumor, or treatment or prevention of infectious diseases. Here, the cancer and malignant tumor are not particularly limited. For example, digestive organ cancer such as colon cancer and gastric cancer, myeloma, liver cancer, leukemia, melanoma, prostate cancer, breast cancer, uterine cancer, lung cancer, oral cavity Examples include cancer and brain tumors. In addition, although not particularly limited, infectious diseases include viral infections such as chronic hepatitis due to hepatitis C and hepatitis C virus, HIV, influenza, and the like.

[0012] The extract of Cordyceps sinensis in the present invention may be an extract of fruit bodies and Z or mycelium. The extract is preferably prepared using an extract of the mycelium of Cordyceps sinensis, particularly preferably in the state of the mycelium in the previous stage of the edible fruit body. Cordyceps sinensis may be produced by culture or collected from nature. For example, mycelium obtained by culturing Cordyceps sinensis bacteria in a solid medium can be used. The extract of Cordyceps sinensis used in the present invention can be prepared by a method known in the art. For example, a solid medium containing mycelium is pulverized and decomposed in the presence of water and an enzyme. The extract obtained in this way can be used. As a solvent used for the preparation of the extract, for example, water, ethanol, methanol, butanol, isopropanol, etc., preferably water can be used. Extraction can be performed under heating of a solvent (for example, about 85 to 105 ° C), but irradiation with ultrasonic waves at a lower temperature (for example, 25 to 50 ° C, preferably 30 to 45 ° C) You can do it from here.

[0013] The Cordyceps sinensis extract thus obtained has a carbohydrate content of 1-50%, preferably 5-20% (wZw) by the carbohydrate analysis by the phenol-sulfuric acid method, and the protein by the protein analysis by the Lowry method. 1 Power including 50%, preferably 5-20% (wZw) It is not limited.

[0014] The Cordyceps sinensis extract in the present invention may be a fraction obtained from the extract. The fractionation method may be a method commonly used in the technical field to which the present invention belongs. Examples of the fractionation method include fractionation by extraction using an arbitrary solvent, gel filtration force, ram chromatography. , Fractionation using an ion exchange column, and silica gel column chromatography. The fractionation method may be one type of method or a combination of a plurality of means. The solvent used in the fractionation method is not particularly limited. For example, water, methanol, ethanol, n propanol, isopropanol, acetic acid, acetone, and mixtures thereof can be used.

[0015] The extract and fraction used in the present invention use, as necessary, a concentrated solution, a viscous substance or a solid obtained by concentration and Z or freeze-drying as a soluble fraction. be able to.

[0016] As used herein, the term "spider cell activator" generally refers to an action that enhances the function of a rod cell by acting on the rod cell, or a rod cell. It means a drug that has the effect of amplifying the amount of the substance expressed in the cell or inside the cell. 12, the production ability of interferon γ or interleukin 1 etc.) can be increased.

[0017] The rod-shaped cell activator of the present invention can be used as an active ingredient of a pharmaceutical composition. The pharmaceutical composition can be used in various dosage forms such as tablets, capsules, powders, granules, pills, solutions, emulsions, suspensions, solutions, spirits, syrups for oral administration. And parenteral preparations such as subcutaneous injections, intravenous injections, intramuscular injections and intraperitoneal injections. However, it is not limited to these. These preparations can be produced by known methods usually used in the preparation process.

[0018] The pharmaceutical composition may contain various commonly used components, for example, one or more pharmaceutically acceptable excipients, disintegrants, diluents, lubricants, and dressings. Flavoring agent, coloring agent, sweetener, flavoring agent, suspending agent, wetting agent, emulsifying agent, dispersing agent, adjuvant, prevention It may include preservatives, buffers, binders, stabilizers, coating agents, and the like. Further, the pharmaceutical composition of the present invention may be a sustained or sustained release dosage form.

[0019] The dosage of the pharmaceutical composition of the present invention can be appropriately selected depending on the administration route, the patient's body shape, age, physical condition, degree of disease, elapsed time after onset, etc. The pharmaceutical composition of the present invention The article can include a therapeutically effective amount and a Z or prophylactically effective amount of a rod cell activator. Cordycebus sinensis mycelium extract was originally used as a food and herbal medicine and has little concern for hyperplasia! / It is a relatively safe substance, so it can be administered at high concentrations as needed It is. In the present invention, Cordyceps extract is generally used at a dose of 100-5000 mgZ day Z adult, preferably 300-3000 mgZ day Z adult. The pharmaceutical composition may be administered in a single dose or multiple doses, for example, other rod-like cell activators, immunostimulators, anticancer agents, antitumor agents, anti-infective agents, etc. It can also be used in combination with drugs.

[0020] According to still another aspect of the present invention, there is provided a method of inducing mature rod cells by treating immature rod cells with the above rod-shaped cell activator. Here, immature immature rod cells are not particularly limited. For example, monocyte cells are cytosylated (for example, condylar granule Z macrophage colony stimulating factor (hereinafter also referred to as “GM-CFS”) and interleukins. 4 (hereinafter also referred to as “IL 4”)). Here, the monocyte cell is not particularly limited. For example, a blood monocyte cell can be used, and a human blood monocyte cell is preferable.

[0021] The change of immature rod cells into mature rod cells can be confirmed by methods known to those skilled in the art in the art. For example, costimulatory factors common to rod cells (for example, CD40, CD80, CD86) and an increase in the expression level of a protein (eg, CD83) specific to mature rod cells can be determined. The expression levels of these proteins can be observed by, for example, a flowmetry method.

[0022] The rod-shaped cell activator of the present invention for use in activating rod-shaped cells in vitro is, in an amount, 10-1000 μgm, preferably 100-500 μgZmL per day. It can be added to the culture medium. Further, for example, the activity of rod-shaped cells can be induced by culturing for a period of 1 to 15 days, preferably 2 to 3 days. [0023] The method of the present invention may further include a step of mixing the rod-shaped cells and the antigenic substance in order to cause the obtained mature rod-shaped cells to present a specific antigen. Here, the antigenic substance is not particularly limited, but tumor-derived peptides or virus-derived peptides can be used, and for example, CEA-derived peptides, MAGE-derived peptides and the like can be used. Mixing of the rod-shaped cells and the antigenic substance may be performed before or after the activation of the rod-shaped cells or simultaneously with the activation of the rod-shaped cells, but is preferably performed simultaneously with the activation of the rod-shaped cells.

[0024] According to yet another aspect of the present invention, a mature rod cell obtained by treating an immature rod cell with a rod cell activator, and a pharmaceutical composition comprising the mature rod cell Is provided. Here, the pharmaceutical composition is not particularly limited, but can be used for preventing or treating cancer, malignant tumors or infectious diseases. For example, digestive organ cancer such as colon cancer and gastric cancer, bone marrow Cancer, liver cancer, leukemia, melanoma, prostate cancer, breast cancer, eclampsia, lung cancer, oral cancer, brain tumor and other cancers or malignant tumors; or chronic hepatitis due to hepatitis B or C virus, HIV, influenza, etc. Can be applied to. The auxiliary activator of the present invention can contain various commonly used components, and the dosage thereof depends on the route of administration, the patient's body shape, age, physical condition, degree of disease, elapsed time after onset, etc. It can be selected appropriately. For example, the auxiliary activator of the present invention can be carried out in the same embodiment as the above-described pharmaceutical composition of the present invention.

[0025] According to a further aspect of the present invention, there is provided an auxiliary activator for use in immunotherapy, comprising the above-described rod-shaped cell activator. Here, the auxiliary activator is a substance that is administered into the living body together with the rod-shaped cells in order to induce the activation of the rod-shaped cells during immunotherapy, and is included as one component of the pharmaceutical composition. Also good. Here, diseases targeted for immunotherapy are not particularly limited. For example, gastrointestinal cancer such as colon cancer and gastric cancer, myeloma, liver cancer, leukemia, melanoma, prostate cancer, breast cancer, uterine cancer, It can be applied to cancers such as lung cancer, oral cancer, brain tumor or malignant tumors; or chronic hepatitis due to hepatitis B or C virus, HIV, influenza and other infections.

[0026] According to still another aspect of the present invention, there is provided a food composition comprising the above-described rod-shaped cell activator. The food composition of the present invention includes a liquid beverage such as a functional beverage. The food The product composition can be used as a functional food, as well as a quasi-drug, a food and drink component, and a food additive. In addition, the food composition in the present specification can be used as a functional food as it is, and can also be used as a food and drink, a drug, a quasi-drug, a component of a food and drink, a food additive and the like. The use enables daily and continuous ingestion of food, beverage, food composition or composition for oral intake having a rod-like cell activation effect and an immunostimulatory effect, and an effective rod-cell activation effect. It is possible to improve the constitution by immunostimulation effect, treat diseases such as cancer, malignant tumors and infectious diseases and prevent their onset. Examples of food compositions, foods or beverages containing the rod-like cell activator or immunostimulatory agent of the present invention include functional foods and health foods having rod-like cell activation effects or immunostimulatory effects. , General foods (juice, confectionery, processed foods, etc.), nutritional supplements (nutrient drinks, etc.). The food or beverage in the present specification includes, but is not limited to, inorganic components such as iron and calcium, various vitamins, dietary fibers such as oligosaccharides and chitosan, proteins such as soybean extract, lipids such as lecithin, It contains saccharides such as sucrose and lactose, and plant extracts such as shiitake mushrooms.

The invention's effect

[0027] As shown in the following examples, the rod-shaped cell activator of the present invention has an action of promoting maturation of immature rod cells and an immunostimulatory action resulting from the action. Therefore, the present invention provides an effective means for treating and / or preventing cancer, malignant tumors and infectious diseases caused by immunostimulation.

Furthermore, the present invention provides a method for inducing mature rod cells by treating immature rod cells. Since the rod cells activated by the method of the present invention have a high IL-12 production ability, they strongly promote the activity of Thl cells through IL 12 production and have a high therapeutic effect on the disease or It is thought to exert a preventive effect.

Example

[0029] In the following, the power for explaining the preferred embodiments of the present invention in more detail The present invention is not limited to these embodiments.

[Example 1] Preparation of Cordyceps extract Cordycebus sinensis extract (hereinafter also referred to as “corticus extract”) was prepared by the following procedure. Cordycebus sinensis mycelium was liquid cultured at 15-18 ° C in a medium obtained by adding glucose solution to corn powder extract, koji powder extract, and soybean powder extract. After culturing, the mycelium was separated by high-speed centrifugation and dried with a laminar flow dryer to obtain Cordyceps sinensis mycelium powder. To 1 OOg of Conoregiceptus sinensis mycelium powder, 2 L of distilled water was added and extracted at 90-95 ° C for 4 hours. The extract was filtered, and the filtrate was concentrated under reduced pressure to obtain Cordyceps extract powder. The recovery rate was about 30% in terms of mycelium powder force conversion.

[Example 2] Measurement of human rod-like cell activation effect of Cordyceps extract

(2-1) Induction of immature rod cells from human peripheral blood

Healthy human peripheral blood was Nono phosphorus bled f a, RosetteSep Humon monocyte Enrichment cocktail kit (Stemcell, Inc.) using monocytes adjust the protocol as the kit, RPMI1640 medium (sigma, 10% FBS-containing) to 5xl0 ⁵ cells The cell suspension was prepared so as to be / ml. 500 L of the prepared cell suspension was dispensed into a 24-well plate, and further added to each of GM-CSF (Peprotech) and IL-4 (Peprotech) at 40 ng / ml. This was cultured at 37 ° C, 5% CO for 3 days. Be careful not to suck the cells after 3 days.

2

Deeply blotted and fresh RPMI 1640 medium (sigma, containing 10% FBS) 300 / z L was added. Further, GM-CSF (Peprotech) and IL-4 (Peprotech) were added at 40 ng / ml, and further cultured for 3 days (6 days in total) to induce immature rod cells. Induction of immature rod cells is a protein-specific antibody that expresses the expression of CD14 protein, a monocyte marker on the cell surface, and the expression of costimulatory factors, CD40, CD80, and CD86. Anti-CD14 antibody FITC labeling, anti-CD40 antibody FITC labeling, anti-CD80 antibody PE labeling, and anti-CD86 antibody PC5 labeling (Peprotech) were measured with a flow cytometer (Beckman Coulter Epics XL) for confirmation.

(2-2) Induction of mature rod cells

To immature rod cells prepared as described above, Cordyceps extract and PSK were added to a final concentration of 200 μg / ml, and cultured at 37 ° C., 5% CO for 2 days. Also, the control

2

The cells were cultured for 2 days at 37 ° C and 5% CO without adding anything. 2 days later, culture under each condition Cell surface costimulatory factors CD40, CD80, CD86 protein and mature rod cell specific marker CD83 protein expression levels of anti-CD40 antibody FITC label, anti-CD80 antibody PE label, anti-CD86 antibody PC5 label ( Measurement was performed with a flow cytometer (Beckman Coulter Epics XL) using Peprotech and anti-CD83 antibody FITC labeling (Peprotech). The results are shown in Figure 1.

[0031] As a result of the measurement, when either Cordyceps extract or PSK was added, the expression level was increased for all markers compared to the control. It was confirmed that dendritic cells were activated and maturation was promoted. In addition, the expression level of Cordyceps extract for all markers exceeded the level of PSK, and in particular, the type added to CD83, a mature rod-shaped cell-specific marker, was added. The expression level in was confirmed to be about 3 times that of the system to which PSK was added. From this, it was suggested that the rod-shaped cell activator of the present invention has a high potency-activating effect as compared with conventionally known rod-shaped cell activators.

[Example 3] Measurement of IL-12-producing ability of human rod-like cells activated by Cordyceps extract In the same manner as in Example 2, immature rod-like cells were induced from human peripheral blood. Cordyceps extract and PSK were added to the prepared immature rod cells so that the final concentration was 200 g / ml, and the cells were cultured at 37 ° C., 5% CO for 24 hours. As a control, 37 ° C without any addition,

2

The cells were cultured for 24 hours under 5% CO conditions. After 24 hours, each culture supernatant is collected and stored in the culture supernatant.

2

The amount of IL-12 was measured with an ELISA kit (Biosource) according to the kit protocol. The result is shown in figure 2.

[0032] In the system supplemented with PSK, there was almost no increase in IL-12 production compared to the control. On the other hand, in the Cordyceps extract system, a significant increase in IL-12 production was confirmed. From this, it was confirmed that the rod-shaped cells activated by Cordyceps extract have a high IL-12 productivity.

Brief Description of Drawings

[0033] [Fig. 1] An example of test results comparing the ability of Cordyceps mycelium extract and PSK to activate rod-shaped cells.

[Fig. 2] Inter-mouth of rod-shaped cells activated by Cordyceps mycelium extract and PSK It is an example of a test result comparing the production capacity of IKIN-12.

Claims

The scope of the claims

[I] A rod-shaped cell activator comprising an extract of Cordyceps sinensis (Berkely) Saccardo.

[2] The rod-shaped cell activator according to claim 1, wherein the extract of Cordycesbus sinensis is a hot water extract of mycelium.

[3] The rod-shaped cell activator according to claim 1 or 2, wherein activation of the rod-shaped cells promotes maturation of the rod-shaped cells.

[4] The rod-shaped cell according to any one of claims 1 to 3, wherein the activity of the rod-shaped cell enhances the ability of the rod-shaped cell to produce interleukin 12, interferon γ or interleukin 1. Activator.

[5] A pharmaceutical composition comprising the rod-shaped cell activator according to any one of claims 1 to 4.

[6] A pharmaceutical composition comprising the rod-like cell activity agent according to any one of claims 1 to 4, which is used for immunostimulation.

[7] A pharmaceutical composition comprising the rod-shaped cell activator according to any one of claims 1 to 4, for treating or preventing cancer or a malignant tumor.

[8] A pharmaceutical composition comprising the rod-shaped cell activator according to any one of claims 1 to 4, for treating or preventing an infectious disease.

[9] A food composition comprising the rod-shaped cell activator according to any one of claims 1 to 4.

[10] A method for inducing mature rod cells by treating immature rod cells with the rod cell activator according to any one of claims 1 to 4.

[I I] A mature rod-shaped cell obtained by treating an immature rod-shaped cell with the rod-shaped cell activator according to any one of claims 1 to 4.

[12] A pharmaceutical composition comprising the mature rod-shaped cell according to claim 11.

[13] The pharmaceutical composition according to claim 12, for preventing or treating cancer, malignant tumor or infectious disease.

[14] An auxiliary activator for use in immunotherapy, comprising the rod-shaped cell activator according to claim 1-14.