CA2352512A1 - Lak activity enhancers derived from extract of lentinus endodes mycelium and lak activity-enhancers formulations containing the extract - Google Patents
Lak activity enhancers derived from extract of lentinus endodes mycelium and lak activity-enhancers formulations containing the extract Download PDFInfo
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Abstract
An immunopotentiator and immunopotentiating preparations which show little side effects and can be obtained economically. An LAK activity potentiator containing a shiitake Cortinellus shiitake mushroom hyphae extract.
Description
SPECIFICATION
LAK ACTI'iIITY ENHANCERS DERIVED FROM
EXTRACT OF LENTINUS EDODES MYCELIUM AND
LAK ACTIVITY-ENHANCING FORMULATIONS CONTAINING THE EXTRACT
FTFT,D OF THE INVENTIWI
The present invention relates to the immunological field. In particular, it relates to immune activity enhancers and therapeutic formulations containing an extract of Lentinus edodes mycelium. And more particularly, it relates to materials useful for enhancing LAK activity and LAK activity-enhancing formulations containing the extract.
pRTOR ART
The activity o~f the immune system can be classified into two modes of action: Cell-mediated immunity and humoral immunity. Humoral immunity refers to an immune response involving antibodies, while cell-mediated immunity refers to an immune response produced by cells acting directly on a target.
Lymphocytes p7Lay the main role in this cell-mediated immune response and often target cell surface substances as antigens. Cell-mediated immunity is thought to be also involved in immune response against tumor, intracellular parasites, and viruses, transplantation, some drug hypersensitivity and some autoimmune diseases. Generally, the reaction is often specific, but can also be non-specific.
For example, iit is known in tumor immunology that tumor cells possess tumor antigens. That is, it is known that therE: are twc> types of antigens, one type consisting of tumor-specific antigens (TSA) and the other of tumor-s associated antigens (TAA) which is also present in normal cells at a very low level, and upregulated with malignant transformation .
There tumor antigens are expressed by alteration in a genetic sE:quence or by mutation of autologous cells. A
common therapy usE:d against tumor cells having abnormal antigenic expression is immunotherapy, including immunization with a tumor antigen or administration of a drug which enhances the immune function. It is generally known that natural killer (NK) cells demonstrate higher tumor cell-destroying activity than normal cells and that immunotherapy can also enhance the activity of NK cells.
NK cells are cytotoxic lymphocytes also present in normal individuals and ar_e known to exhibit MHC antigen-nonrestricted cytotoxicity against tumor cells, virus-infected .cells or the like. However, it has been recently shown that tumor cells exist which are resistant to even NK
cells.
Dr. S. Rosenberg of the United States National Cancer Institute (NCI) found that incubation of peripheral lymphocytes with .int:erleukin 2 (IL-2) can induce killer cells showing cytotoxicity to a wide range of target cancer cells, including aut:ologous cancer cells, and that these killer cells can k:i.7_1 even NK cell-resistant cancer cells (see Japanese patent public disclosure No. 116518/87).
These killer cells were named lymphokine activated killer ( LAK ) cell.s . LAK ce:L.ls do not consist of a cytologically homogeneous population and are known to include NK cells and killer T-cells.
Recently, adopi~ive immunity has been attempted wherein peripheral. lymphocytes from a patient are activated with IL-2 in a cell culture system, and then LAK cells showing antitumor activity are reinfused into the patient (LAK therapy). It: has been reported that remission from terminal <:ancer has been achieved or tumor growth-inhibited by the use of adoptive immunotherapy involving repeated administration of such LAK cells. However, IL-2-mediated LAK therapy involves a number of problems such as the physical stress associated with the isolation of a great number of leukocytes, the high cost of performing mass culture o:f isolated leukocytes, or the use of expensive IL-2, etc, a:nd, moreover, IL-2 administration often causes serious side effects.
Specifically, i.t is known that LAK adoptive immunotherapy using IL-2 causes side effects such as general prostration, chills, fever, hypoalbuminemia, anemia, eosinophilia and that these side effects are more serious than those caused by administration of IL-2 alone. More notably, some important side effects are strongly associated with the cytotoxicity of LAK cells against normal cells. It is also reported that such cytotoxicity of LAK cells against hematopoietic stem cells induces anemia and thrombocytopenia, as well as causing in vitro damages to lymphocytes, macrophages and vascular endothelial cells. Moreover, IL-2 administered via the oral route: is poor~_y absorbed and must therefore mainly be administered via injection and direct administration at the present tame.
Thus, there is a demand for the development of therapeutic agents capable of overcoming these disadvantages, especially agents that could enhance the LAK
activity of lymphocytes without using IL-2 or pharmaceutical agents containing such agents.
Anticancer agents are generally targeted at abnormal growth of cancer cells and can be classified by target into nucleic acid biosynthesis inhibitors such as alkylating agents, nucleic acidL biosynthesis substrate analogs, antibiotics, steroid hormones, etc. and mitotic inhibitors such as plant alkaloids.
However, such anticancer agents also cause noticeable side effects with rE:gard to normal proliferative cells such as bone marrow, gastrointestinal epithelium, and hair follicle. That is, they generally cause nausea, vomiting, ulcers of the mouth and small bowel, diarrhea, epilation, bone marrow inhibition leading to underproduction of major blood components, and so on, irrespective of the administration routf;.
Some bacteria,. foods and other naturally occurring substances are known to have anticancer properties.
Bacteria and food-tye substance are generally far preferential for use as anti-cancer agents due to their generally benign nature and low side-effect profile. Many attempts have been made to cure cancer by using bacteria, as shown in reports relating to Coley's toxin consisting of a culture filtrate o:f Serratia marcesens and Streptococcus pyogenes (1964); treatment of leukemia with BCG (Mathe, G., Adv. CancE:r Res., 14, 1, 1971); tumor regression in guinea pigs (Zbar, B., et: al., J. Natl. Cancer Inst., 48, 831, 1971); an<i effectiveness of administration of yeast cell wall polysaccharide against transplanted tumor cells such as sarcoma 180, for example, Especially, a great amount of research has been conducted into thE: anticancer effect of polysaccharides derived from yeast such as yeast glucan and yeast mannan, from other bactei~~um, from lichens and from basidiomycetes.
Among them, commercial products currently available on the market as anticancer immunopotentiators include Krestin derived from the cultured mycelia of kawaratake (Coriolus versicolor, Basidiomycetes: Polyporaceae) (booster of immune function o:f hosts, Kureha Chemical Industry and Sankyo Co.Ltd.), a polysaccharide derived from shiitake (Lentinus edodes) called lentinan and a polysaccharide derived from suehirotake (Schizophyllum Commune).
Lentinus edode;s (Shiitake) is a common edible mushroom found in both Japan and China, and has been cultivated in Japan for around 300 years. It has been recently elucidated for its pharmacological effects and effective: ingredients and reported to have various effects, such as growth inhi.b:ition effects on transplanted tumor cells in t:he large bowel and liver in rats and mice (Sugano N. et al., Cancer Letter, 27:1, 1985; Suzuki Y. et al., Journal of. the Japan Society of Coloproctology, 43:178, 1990 ) ; mit:ogenic e:f:f~ect ( Tabata T . et al . , Immunophar_macology, 24:57, 1992; Hibino et al., Immunopharmacology,. 28:77, 1994), etc.
nTg L OSURE OF THE ~VENTION
The present inventors researched the immunopotentiatinc~ activity, antitumor activity and/or anticancer activit=y of Lentinus edodes with a view to providing an inexpensive immunotherapeutic agent having few side effects.
The present invention was accomplished on the basis of the finding that an extract of the mycelium, which is a precursor to the edible fruiting body of Lentinus edodes, has a far higher :immmnopotentiating activity, antitumor activity and/or anticancer activity than the fruiting body and that said extracts also has an LAK activity-enhancing activity.
According to the first aspect of the present invention, therefore, an LAK activity enhancer containing an extract of Lentir~us edodes mycelium is provided.
"LA:K activity" refers to the cytotoxic activity of cytotoxic T-lymphocytes, which attacks tumors unrecognizable by .lymphocytes having NK activity but which have little influence on autologous normal cells. "LAK
LAK ACTI'iIITY ENHANCERS DERIVED FROM
EXTRACT OF LENTINUS EDODES MYCELIUM AND
LAK ACTIVITY-ENHANCING FORMULATIONS CONTAINING THE EXTRACT
FTFT,D OF THE INVENTIWI
The present invention relates to the immunological field. In particular, it relates to immune activity enhancers and therapeutic formulations containing an extract of Lentinus edodes mycelium. And more particularly, it relates to materials useful for enhancing LAK activity and LAK activity-enhancing formulations containing the extract.
pRTOR ART
The activity o~f the immune system can be classified into two modes of action: Cell-mediated immunity and humoral immunity. Humoral immunity refers to an immune response involving antibodies, while cell-mediated immunity refers to an immune response produced by cells acting directly on a target.
Lymphocytes p7Lay the main role in this cell-mediated immune response and often target cell surface substances as antigens. Cell-mediated immunity is thought to be also involved in immune response against tumor, intracellular parasites, and viruses, transplantation, some drug hypersensitivity and some autoimmune diseases. Generally, the reaction is often specific, but can also be non-specific.
For example, iit is known in tumor immunology that tumor cells possess tumor antigens. That is, it is known that therE: are twc> types of antigens, one type consisting of tumor-specific antigens (TSA) and the other of tumor-s associated antigens (TAA) which is also present in normal cells at a very low level, and upregulated with malignant transformation .
There tumor antigens are expressed by alteration in a genetic sE:quence or by mutation of autologous cells. A
common therapy usE:d against tumor cells having abnormal antigenic expression is immunotherapy, including immunization with a tumor antigen or administration of a drug which enhances the immune function. It is generally known that natural killer (NK) cells demonstrate higher tumor cell-destroying activity than normal cells and that immunotherapy can also enhance the activity of NK cells.
NK cells are cytotoxic lymphocytes also present in normal individuals and ar_e known to exhibit MHC antigen-nonrestricted cytotoxicity against tumor cells, virus-infected .cells or the like. However, it has been recently shown that tumor cells exist which are resistant to even NK
cells.
Dr. S. Rosenberg of the United States National Cancer Institute (NCI) found that incubation of peripheral lymphocytes with .int:erleukin 2 (IL-2) can induce killer cells showing cytotoxicity to a wide range of target cancer cells, including aut:ologous cancer cells, and that these killer cells can k:i.7_1 even NK cell-resistant cancer cells (see Japanese patent public disclosure No. 116518/87).
These killer cells were named lymphokine activated killer ( LAK ) cell.s . LAK ce:L.ls do not consist of a cytologically homogeneous population and are known to include NK cells and killer T-cells.
Recently, adopi~ive immunity has been attempted wherein peripheral. lymphocytes from a patient are activated with IL-2 in a cell culture system, and then LAK cells showing antitumor activity are reinfused into the patient (LAK therapy). It: has been reported that remission from terminal <:ancer has been achieved or tumor growth-inhibited by the use of adoptive immunotherapy involving repeated administration of such LAK cells. However, IL-2-mediated LAK therapy involves a number of problems such as the physical stress associated with the isolation of a great number of leukocytes, the high cost of performing mass culture o:f isolated leukocytes, or the use of expensive IL-2, etc, a:nd, moreover, IL-2 administration often causes serious side effects.
Specifically, i.t is known that LAK adoptive immunotherapy using IL-2 causes side effects such as general prostration, chills, fever, hypoalbuminemia, anemia, eosinophilia and that these side effects are more serious than those caused by administration of IL-2 alone. More notably, some important side effects are strongly associated with the cytotoxicity of LAK cells against normal cells. It is also reported that such cytotoxicity of LAK cells against hematopoietic stem cells induces anemia and thrombocytopenia, as well as causing in vitro damages to lymphocytes, macrophages and vascular endothelial cells. Moreover, IL-2 administered via the oral route: is poor~_y absorbed and must therefore mainly be administered via injection and direct administration at the present tame.
Thus, there is a demand for the development of therapeutic agents capable of overcoming these disadvantages, especially agents that could enhance the LAK
activity of lymphocytes without using IL-2 or pharmaceutical agents containing such agents.
Anticancer agents are generally targeted at abnormal growth of cancer cells and can be classified by target into nucleic acid biosynthesis inhibitors such as alkylating agents, nucleic acidL biosynthesis substrate analogs, antibiotics, steroid hormones, etc. and mitotic inhibitors such as plant alkaloids.
However, such anticancer agents also cause noticeable side effects with rE:gard to normal proliferative cells such as bone marrow, gastrointestinal epithelium, and hair follicle. That is, they generally cause nausea, vomiting, ulcers of the mouth and small bowel, diarrhea, epilation, bone marrow inhibition leading to underproduction of major blood components, and so on, irrespective of the administration routf;.
Some bacteria,. foods and other naturally occurring substances are known to have anticancer properties.
Bacteria and food-tye substance are generally far preferential for use as anti-cancer agents due to their generally benign nature and low side-effect profile. Many attempts have been made to cure cancer by using bacteria, as shown in reports relating to Coley's toxin consisting of a culture filtrate o:f Serratia marcesens and Streptococcus pyogenes (1964); treatment of leukemia with BCG (Mathe, G., Adv. CancE:r Res., 14, 1, 1971); tumor regression in guinea pigs (Zbar, B., et: al., J. Natl. Cancer Inst., 48, 831, 1971); an<i effectiveness of administration of yeast cell wall polysaccharide against transplanted tumor cells such as sarcoma 180, for example, Especially, a great amount of research has been conducted into thE: anticancer effect of polysaccharides derived from yeast such as yeast glucan and yeast mannan, from other bactei~~um, from lichens and from basidiomycetes.
Among them, commercial products currently available on the market as anticancer immunopotentiators include Krestin derived from the cultured mycelia of kawaratake (Coriolus versicolor, Basidiomycetes: Polyporaceae) (booster of immune function o:f hosts, Kureha Chemical Industry and Sankyo Co.Ltd.), a polysaccharide derived from shiitake (Lentinus edodes) called lentinan and a polysaccharide derived from suehirotake (Schizophyllum Commune).
Lentinus edode;s (Shiitake) is a common edible mushroom found in both Japan and China, and has been cultivated in Japan for around 300 years. It has been recently elucidated for its pharmacological effects and effective: ingredients and reported to have various effects, such as growth inhi.b:ition effects on transplanted tumor cells in t:he large bowel and liver in rats and mice (Sugano N. et al., Cancer Letter, 27:1, 1985; Suzuki Y. et al., Journal of. the Japan Society of Coloproctology, 43:178, 1990 ) ; mit:ogenic e:f:f~ect ( Tabata T . et al . , Immunophar_macology, 24:57, 1992; Hibino et al., Immunopharmacology,. 28:77, 1994), etc.
nTg L OSURE OF THE ~VENTION
The present inventors researched the immunopotentiatinc~ activity, antitumor activity and/or anticancer activit=y of Lentinus edodes with a view to providing an inexpensive immunotherapeutic agent having few side effects.
The present invention was accomplished on the basis of the finding that an extract of the mycelium, which is a precursor to the edible fruiting body of Lentinus edodes, has a far higher :immmnopotentiating activity, antitumor activity and/or anticancer activity than the fruiting body and that said extracts also has an LAK activity-enhancing activity.
According to the first aspect of the present invention, therefore, an LAK activity enhancer containing an extract of Lentir~us edodes mycelium is provided.
"LA:K activity" refers to the cytotoxic activity of cytotoxic T-lymphocytes, which attacks tumors unrecognizable by .lymphocytes having NK activity but which have little influence on autologous normal cells. "LAK
activity-Enhancing" refers to the effect of enhancing this LAK activity, that: is directly or indirectly inducing the production of LAK cells from lymphocytes or further enhancing the activity of existing LAK cells.
Enhancement of LAK activity increases antitumor activity of LAK cells, which leads to an improvement in the function of the cell-mediated immune system. Thus, the present invention can be applied not only to treatments for improving antitumor activity but also to treatments for improving the immune function.
Lymphocytes derived from peripheral blood may be treated with LAK activity enhancers of the present invention to enhance: LAK activity. They may directly act on lymphocytes derived from peripheral blood preferably at an extract of Lent inus edodes mycelium concentration of 1 ~g or more per 10'' lymphocytes or 1 ~g or more per 1 ml of peripheral blood. LAK activity enhancers of the present invention may be used in LAK therapy in place of IL-2.
LAK activity e:nhancers of the present invention may consist of an extract of Lentinus edodes mycelium on its own or may further contain materials other than an extract of Lentinus edodes nnycelium. The range of such materials is not limited so long as the LAK activity enhancing-activity of the extract of Lentinus edodes mycelium is not affected by their u:~e.
LAK activity E:nhancers of the present invention may also be combined with other materials having an immunopot:entiating activity and antitumor activity and/or antlCancel_' aCtlvlt:y.
According to the second aspect of the present invention,, an LAK activity-enhancing formulation comprising an LAK activity enhancer containing an extract of Lentinus edodes mycelium is provided.
LAK activity-enhancing formulations of the present invention may be i~he extract of_ Lentinus edodes mycelium itself or pharmaceutical or veterinary formulations comprising an LAK activity enhancer containing an extract of Lentinus edodes mycelium and a pharmaceutically acceptable carrier.
LAK activity-enhancing formulations of the present invention may be suitable for oral administration or injection or percutaneous administration.
LAK activit~~-enhancing formulations of the present invention may be in the form of a food, drink or feed.
LAK activity-enhancing formulations of the present invention may be used for treating tumor and/or cancer.
LAK activity-enhancing formulations of the present invention do not spE:cifically attack specific tumor cells, because they induce LAK cells. Therefore, LAK activity-enhancing formulations of the present invention can be used for treating various tumors, either epithelial or non-epithelial.
LAK: activity-enhancing formulations of the present invention may also 'be used for treating other diseases than tumor or cancer, such as the improvement of the immune function,. Specifically, LAK activity-enhancing _ g _ formulations of th.e present invention may also be used for the improvement of immune function compromised by immunocompromising therapies or accidents such as radiation, autoimmune diseases, infectious diseases, etc.
LAK activity-enhancing formulations of the present invention can also be used as prophylactic agents against various d_Lseases. For example, they may be administered to prevent tumor and/or cancer or to prevent bacterial or viral infections.
LAK activity-enhancing formulations of the present invention may be used in combination with other drugs such as antitumor and/or anticancer agents such as immunopotentiators, antibiotics, analgesics, antiinf lammatory age:r~ts , etc .
According to the third aspect of the present invention, a method for treating tumor and/or cancer comprising administE:ring an effective amount of said LAK
activity-enhancing formulation is provided.
The method for treating tumor and/or cancer of the present invention may be used in combination with other therapies such as oither chemotherapy, surgical treatment, radiation, thermotherapy, etc.
According to i~he fourth aspect of the present invention, a use of an LAK activity enhancer for obtaining a drug used for t:re,ating tumor and/or cancer is provided.
In order to obtain a drug used for treating tumor and/or cancer, LAK activity enhancers of the present invention may be used in combination with other materials having an immunopotentiating activity and antitumor activity and/or anticancer activity.
BRIEF DESCRIPTION ~' THE DRAWINGS
FIG. 1 is a bar graph showing the LAK activity-enhancing effect of the extract of Lentinus edodes mycelium directly added to peripheral blood as in comparison with IL-2'..
FIG. 2 is a bar graph showing the change of LAK
activity with the concentration of the extract of Lentinus edodes mycelium directly added to peripheral blood.
FIG. 3 is a ba.r graph showing the LAK activity obtained by directly adding Krestin to peripheral blood.
FIG. 4 is a ba.r graph showing the LAK activity in peripheral blood before and after oral administration of the extract of Lentinus edodes mycelium.
An extract of Lentinus edodes mycelium contained in LAK activity enhanc~:rs of the present invention refers to an extract obtained by crushing and decomposing mycelia grown from Lentinus edodes cultured on a solid medium, or a solid medium itself containing Lentinus edodes mycelia in the presence of water and an enzyme.
An extract of Lentinus edodes mycelium used herein is preferably obtained by, but not specifically limited to, the following process.
Lentinus edodE~s spawn is inoculated on a solid medium based on bagasse (sugar cane residue) and defatted rice bran to grow mycelia, and then the solid medium containing the grown mycelia is delignified to enable about 30~ by weight or less to pass through a 12-mesh sieve.
To this delign:ified solid medium are added water and one or more enzymes selected from cellulase, protease or glucosidase while maintaining said solid medium at a temperature of around 30-55°C, and said solid medium is crushed and ground in the presence of said enzyme so that at least '70~ by weight of bagasse fiber is able to pass through a 12-mesh sieve. Then, the temperature is raised to 80-100"C to ensure inactivation of the enzyme and sterilization, and the resulting suspension is filtered to give an extract of Lentinus edodes mycelium.
The extract of Lentinus edodes mycelium as prepared above may be directly used in LAK activity enhancers or LAK
activity-enhancing farmulations of the present invention, but conveniently concentrated and freeze-dried into powder to be stored and used in various forms. The freeze-dried product is a brown powder with hygroscopic characteristics and has a peculiar taste and odor.
LAK activity e:nhancers containing the prepared extract of Lentinus edodes mycelium (or the extract of Lentinus edodes mycE:lium on its own) may be used in LAK
therapy in place of IL-2 to enhance the immune activity against tumor of thE: subject .
LAK therapy gE:nerally comprises the steps of culturing in vitro :Lymphocytes from the subject with IL-2 to induce LAK cells from the lymphocytes, and reinfusing the LAK cells into the subject. At the step of reinfusing LAK cells into thE: subject, IL-2 may be (directly) cocurrent:Ly administered.
LAK activity enhancers cantaining the extract of Lentinus edodes mycelium of the present invention may be cultured :in vitro with lymphocytes from the subject (human, animals, etc.) to :induce LAK cells or may be (directly) cocurrently administered to the subject at the step of reinfusing the cu:Ltu.red lymphocytes to the subject. They may be used in combination with other antitumor and/or anticancer agents.
LAK activity enhancers containing the extract of Lentinus edodes mycelium of the present invention may be directly added to peripheral blood collected from the subject, but preferably sterilized with acetone before being added.
LAK activity e:nhancers containing the extract of Lentinus edodes mycE:lium of the present invention can be directly added to pE:ripheral blood collected from the subject preferably at a concentration expressed as the extract o~f Lentinus edodes mycelium of 1 ~g - 1 mg/106 lymphocytes, more preferably 10 ~g - 100 ~g/106 lymphocytes, most preferably 10 Ecg - 50 ~g/106 lymphocytes. The dose per ml of periphera:L blood, which partially depends on the level of lymphocytes in peripheral blood, approximately represents 1 ~g - 2.5 mg, preferably 10 ~.g - 250 dug, more preferab7_y 10 ~.g - 125 ~.g.
LAK activity-~e:nhancing formulations comprising an LAK
activity enhancer containing the extract of Lentinus edodes mycelium of the present invention can be directly administered to the subject to enhance the anti-tumor immune activity oi_' the subject, thereby providing an effect comparable to the effect elicited by LAK therapy using IL-2.
Induction of L.AK cells in the subject such as a human or animals by directly administering the LAK activity enhancers of the present invention may make it possible to reduce physical stress associated with the isolation of a great number of leukocytes and the high cost resulting from producing mass culture. In addition, the use of LAK
activity-enhancers o~f the present invention can also reduce the risk of contamination during the isolation of blood from the subject and. reinfusion can also be reduced.
LAK activity-enhancing formulations of the present invention are administered most preferably, but are not limited to, the oral. route in view of the least stress imposed on the su~bje:ct and the like. They may also be administered via intravenous, intracutaneous, subcutaneous or intramuscular injjection, or percutaneous, nasal, enteral or other routes.
Suitable dosage forms of LAK activity-enhancing formulations of the present invention include, but are not limited to, tablets, capsules, powders, granules, solutions, syrups, injections, creams, ointments, patches, sprays, suppositories, etc.
Pharmaceutically acceptable carriers that can be optionally mixed witlh LAK activity-enhancing formulations of the present invention include, but not limited to, excipients such as lactose, dextrose, starch, crystalline cellulose; binders such as starch, gelatin, methyl cellulose" polyvinylpyrrolidone; disintegrators such as starch, c<~lcium carboxymethylcellulose, carboxymethyl starch; lubricants such as talc, stearates; coating agents such as sucrose, t=alc, gelatin; as well as various brighteners, flavoring agents, colorants, corrigents, solubilizers, stabilizers, suspending agents, absorbefacients or the like known in the art depending on the purpose. For use as injections, various diluents commonly used in this field of art such as water or ethyl alcohol can be used.
The route oi: administration, dose, frequency or others of LAK act:ivi.ty-enhancing formulations of the present invention are determined taking into account the age, weight, condition and other factors of the subject.
The extract of Lentinus edodes mycelium contained in LAK activity-enhancing formulations of the present invention is highly safe as has been traditionally ingested as a component of food. Therefore, the dose is not be strictly limited. For example, the extract of Lentinus edodes mycelium is normally administered preferably at a dose of 100 mg - 10000 mg 2-3 times daily, more preferably 500 mg - 5000 mg three times daily, most preferably 1000 mg - 1500 mg three times daily. It may be administered in combination with other drugs such as antitumor and/or anticancer. agents such as immunopotentiators, antibiotics, analgesics , antiinf l~ammatory agents , etc .
LAK activity-enhancing formulations of the present invention can also be provided in the form of a food.
Preferred forms of food include powders, granules, pastes, jellies, Eac. Granules desirably are supplemented with sugars such as lactose to add a sweet taste. LAK activity-enhancing formulations of the present invention can also be provided _Ln the form of a drink. These foods or drinks may be supplernented with vitamins, minerals such as calcium, alcohols, deodorants such as polyphenols in addition to the extract of Lentinus edodes mycelium. These foods or drinks may include the categories of specific health foods, medical foods or the like.
LAK activity-enhancing formulations of the present invention can also b~e provided in the form of a feed or feed additive. LAK activity-enhancing formulations of the present invention cam be used as a feed or feed additive for domestic animals; to treat and/or prevent tumor occurring in the domestic animals or to treat and/or prevent bacterial ox' viral infections in domestic animals.
As a result, the amount of therapeutic agents such as antibiotics currently used can be reduced, thereby reducing farming costs. Another advantage is that the period during which shipment of animals is suspended due to the administration of antibiotics can be shortened.
LAK activity enhancement with LAK activity enhancers of the present invention can be confirmed by, but not limited to, the pracedure below according to the method of Takagi et al. (Cli.nical Immunology, 19:245-249, 1987).
T~AK activity tes LAK activity can be determined by S~Cr release assay, [3HJ uridine assay or the like. In terms of convenience and objectivity, the SlCr release assay is preferably used.
The SlCr releasE; assay is one of methods for in vitro determining the cytotoxicity against target cells of LAK
cells induced from lymphocytes treated with an LAK activity enhancer, and comprises the steps of:
(i) adding SlCr-labeled sodium chromate to the target cells to label th<: target cells, (ii) reacting the labeled target cells with induced LAK cells, and (ii:i) measuring the amount of SlCr released into the cell culture supernatant from the target cells bursted by the LAK cells.
Subcultured cells used as target cells in the 5lCr release assay are preferably Daudi cells or Raji cells.
Target cells culturE:d in a culture flask or the like are collected by centric=ugation, and labeled with 5lCr via incubation in a 5~ (:OZ incubator at 37°C for 1-2 hours after 100-150 ~Ci 5lCr labeled-sodium chromate has been added. Culture media for target cells are those suitable for the growth of the cells used, and include, for example, liquid media such a;s RPMI 1640 or F-12 appropriately supplemented with serum, antibiotics, etc.
Cultured target cells are washed with PBS three times, and then suspended in a culture medium at a density of 1 x 106 cells/ml to serv<, for assay.
To induce LAK cells, lymphocytes are isolated from peripheral blood of the subject. Heparin is added to peripheral blood from the subject, and monocytes at the interface are separated by density gradient centrifugation on Ficoll~-Conray solution (s. g. - 1.077). The separated monocytes are washed with PBS (pH 7.4, without Ca and Mg) 2-3 times and then suspended in a culture medium (preferab:ly, RPMI :1640 medium (Gibco) containing FBS
(inactivated fetal bovine serum) and/or antibiotics, if desired) at a density of 1 x 106 cells /ml. This suspension is transferred to a Petri dish which has been precoated with autoserum (plasma) at 37°C for 15 minutes, and incubated at :37°C for 1 hour. Unattached cells are recovered as lymphocyte fractions.
The lymphocytes as prepared above are suspended in a culture medium at a predetermined concentration, preferably 1 x 105-106 cells/ml.
The extract of Lentinus edodes mycelium is added to the medium as used f:or preparing the lymphocyte-suspending solution at a final concentration of about 1-100 ~.g/ml.
Predetermined amounts of the lymphocyte-suspending solution and extract:-containing medium are added to wells of a culture plate. In parallel, predetermined amounts of the lymphocyte-floai~ing solution and the medium not containing the extract of Lentinus edodes mycelium are added to wells as an extract-free control. Then, the culture p7_ate is statically incubated in a COz incubator at room temperature for 3 days.
When a 96-we'll U-bottom microtest plate is used, for example, 100 ~1 of t:he lymphocyte-floating solution is preferably added t:o each well at a density of 1 x 10' to 1 x 105 cells/well. The number of cells per well can be appropriately determined by those skilled in the art in accordance with the well volume, the activity of LAK cells, the sensitivity of target cells to LAK cells, etc.
Thus, lymphocytes derived from the subject are cultured :in the presence of the extract of Lentinus edodes mycelium of the present invention at various concentrations (including zero) to induce effector cells. As used herein, the term effector cells refers to cells subjected to said culture treatment far 3 days and includes both lymphocytes cocultured with an L~AK activity enhancer (lymphocytes treated with an LAK activity enhancer) and lymphocytes cultured in the medium alone without LAK activity enhancer (lymphocytes treated under the extract-free condition).
Cultured lymphocytes (effector cells) are recovered from the well and rE:suspended in fresh RPMI 1640 medium containing 10~ FBS ( 1 x 106 cel7_s /ml ) .
In the assay for determining cytotoxicity, each well containing said target cells is further supplied with a predetermined amouni~ of the floating solution containing said various effector cells (lymphocytes treated with LAK
active enhancer or those under the extract-free condition) at a density of 1 x 105 - 1 x 106 cells/ml for experimental dissociation. 1N HC1 in an amount equal to-the effector-floating solution for maximum dissociation, or the medium alone in an amount equal to the effector-floating solution S for natural dissociation. Then, cells are collected at the bottom of the well of the cultuze plate using a plate centrifuge or the like, and then incubated in a S% CO
incubator at 3'7'C fo:r a predetermined period (for example, 2-5 hours:l.
After completion of the incubation, the radioactivity of S~Cr released in i:he culture supernatant in the culture supernatant in each well is measured using a scintillation counter or the like_ The term "maximum dissociation" refers to the total amount of S'Cr relea;:ed when all the target cells are destroyed. Maximum dissociation can be determined by, for example, lysing targret cells via incubation in a culture medium containing 1N HC1 under predetermined conditions in the absence of effector cells and measuring the amount of SlCr released in the culture supernatant.
The term "experimental dissociation" refers to the amount of S=Cr releaaed by cytotoxicity of effector cells against target cells and natural death of target cells when effector cells are added to target cells. Experimental dissociation can be determined by, for example, incubating target cells under the same conditions as those for maximum dissociation with the exception of adding effector cells in place of 1N HC1 to the culture medium and measuring the amount of SlCr released in the culture supernatant.
The term "natural dissociation" refers to the amount of SlCr released by natural death of target cells. Natural dissociation can be determined by, for example, incubating target cells under the same conditions as for maximum dissociation with exception of containing neither 1N HCl nor effector cells and measuring the amount of SlCr released :in the culture supernatant.
The activation level of LAK cells is evaluated on the basis of 'the LAK activity calculated by equation (1) below.
Experimental dissociation(cpm)-Natural dissociation(cpm) LAK activity _ ---- x 100 Maximum dissociation(cpm)-Natural dissociation(cpm) Equation (1) The disclosure of JPA Nos. 353927/98 and 353928/98 on which the present application is based to claim the priority are incorporated herein as a whole as reference.
The following examples further illustrate the present invention but should. not be taken as limiting the scope of the invention thereto. It will be appreciated by those skilled in the art that various changes and modifications can be made without departing from the spirit of the present invention.
E3~AMPLES
A solid med:ium~ consisting of 90 parts by weight of bagasse and 10 parts by weight of rice bran was soaked with an appropriate amount of pure water, and then inoculated with Lentinus edodes spawn and allowed to stand in an incubator at controlled temperature and humidity to grow mycelia. After mycelia spread over the solid medium, the bagasse base was delignified to enable 24~ by weight or less eto pass through a 12-mesh sieve. To 1.0 kg of this delignified medium were added 3.5 L of pure water and 2.0 g of purified cellulase while maintaining the solid medium at 40°C to prepare a medium-containing mixture.
Then, the medium-containing mixture was circulated by a variable speed gear pump, during which the solid medium was crushed and ground at the gears for about 200 minutes so that about 80~ by weight of bagasse fiber is able to pass through a 12~-me,sh sieve. The medium-containing mixture was crushed and ground while the temperature of said mixture was gradually increased. Then, the medium-containing mixture was further heated to 90°C to ensure inactivation of the enzyme and sterilization and allowed to stand at 90°C for :30 minutes. The resulting medium-containing mixture was filtered through a 60-mesh filter cloth to give an extract of Lentinus edodes mycelium solution, which was concentrated and then converted into a freeze-dried powder.
The extract of Lentinus edodes mycelium as prepared above contained 25.:3 (w/w) carbohydrates determined by the phenol-sulfuric acid method, 19.7 (w/w) proteins determined by the Lowry method and 2.6~ (w/w) polyphenols determined by the F'olin-Denis method using gallic acid as standard. The extract of Lentinus edodes mycelium further contains 8~ crude fat, 22~ crude ash and about 20~ soluble nitrogen-free matE:rials other than carbohydrates.
The extract of Lentinus edodes mycelium had a sugar composition (~) as follows: Xyl 15.2, Ara 8.2, Man 8.4, Gul 39.4, Gal 5.4, GlcN 12.0, GluUA 11.3.
Exam In a 2~ Comparison of LAK activities of the extract of r.Pnt; n_ms edodes myce~.? um and IL-2 The extract of Lentinus edodes mycelium obtained in Example 1 was directly administred to peripheral blood collected from the subject to compare the LAK activity-enhancing effect with that of IL-2.
Heparin was added to peripheral blood from subject A, and monocytes at the:.interface were separated by density gradient centrifugation on Ficoll-Conray solution (s.g. -1.077). The separated monocytes were washed with PBS (pH
Enhancement of LAK activity increases antitumor activity of LAK cells, which leads to an improvement in the function of the cell-mediated immune system. Thus, the present invention can be applied not only to treatments for improving antitumor activity but also to treatments for improving the immune function.
Lymphocytes derived from peripheral blood may be treated with LAK activity enhancers of the present invention to enhance: LAK activity. They may directly act on lymphocytes derived from peripheral blood preferably at an extract of Lent inus edodes mycelium concentration of 1 ~g or more per 10'' lymphocytes or 1 ~g or more per 1 ml of peripheral blood. LAK activity enhancers of the present invention may be used in LAK therapy in place of IL-2.
LAK activity e:nhancers of the present invention may consist of an extract of Lentinus edodes mycelium on its own or may further contain materials other than an extract of Lentinus edodes nnycelium. The range of such materials is not limited so long as the LAK activity enhancing-activity of the extract of Lentinus edodes mycelium is not affected by their u:~e.
LAK activity E:nhancers of the present invention may also be combined with other materials having an immunopot:entiating activity and antitumor activity and/or antlCancel_' aCtlvlt:y.
According to the second aspect of the present invention,, an LAK activity-enhancing formulation comprising an LAK activity enhancer containing an extract of Lentinus edodes mycelium is provided.
LAK activity-enhancing formulations of the present invention may be i~he extract of_ Lentinus edodes mycelium itself or pharmaceutical or veterinary formulations comprising an LAK activity enhancer containing an extract of Lentinus edodes mycelium and a pharmaceutically acceptable carrier.
LAK activity-enhancing formulations of the present invention may be suitable for oral administration or injection or percutaneous administration.
LAK activit~~-enhancing formulations of the present invention may be in the form of a food, drink or feed.
LAK activity-enhancing formulations of the present invention may be used for treating tumor and/or cancer.
LAK activity-enhancing formulations of the present invention do not spE:cifically attack specific tumor cells, because they induce LAK cells. Therefore, LAK activity-enhancing formulations of the present invention can be used for treating various tumors, either epithelial or non-epithelial.
LAK: activity-enhancing formulations of the present invention may also 'be used for treating other diseases than tumor or cancer, such as the improvement of the immune function,. Specifically, LAK activity-enhancing _ g _ formulations of th.e present invention may also be used for the improvement of immune function compromised by immunocompromising therapies or accidents such as radiation, autoimmune diseases, infectious diseases, etc.
LAK activity-enhancing formulations of the present invention can also be used as prophylactic agents against various d_Lseases. For example, they may be administered to prevent tumor and/or cancer or to prevent bacterial or viral infections.
LAK activity-enhancing formulations of the present invention may be used in combination with other drugs such as antitumor and/or anticancer agents such as immunopotentiators, antibiotics, analgesics, antiinf lammatory age:r~ts , etc .
According to the third aspect of the present invention, a method for treating tumor and/or cancer comprising administE:ring an effective amount of said LAK
activity-enhancing formulation is provided.
The method for treating tumor and/or cancer of the present invention may be used in combination with other therapies such as oither chemotherapy, surgical treatment, radiation, thermotherapy, etc.
According to i~he fourth aspect of the present invention, a use of an LAK activity enhancer for obtaining a drug used for t:re,ating tumor and/or cancer is provided.
In order to obtain a drug used for treating tumor and/or cancer, LAK activity enhancers of the present invention may be used in combination with other materials having an immunopotentiating activity and antitumor activity and/or anticancer activity.
BRIEF DESCRIPTION ~' THE DRAWINGS
FIG. 1 is a bar graph showing the LAK activity-enhancing effect of the extract of Lentinus edodes mycelium directly added to peripheral blood as in comparison with IL-2'..
FIG. 2 is a bar graph showing the change of LAK
activity with the concentration of the extract of Lentinus edodes mycelium directly added to peripheral blood.
FIG. 3 is a ba.r graph showing the LAK activity obtained by directly adding Krestin to peripheral blood.
FIG. 4 is a ba.r graph showing the LAK activity in peripheral blood before and after oral administration of the extract of Lentinus edodes mycelium.
An extract of Lentinus edodes mycelium contained in LAK activity enhanc~:rs of the present invention refers to an extract obtained by crushing and decomposing mycelia grown from Lentinus edodes cultured on a solid medium, or a solid medium itself containing Lentinus edodes mycelia in the presence of water and an enzyme.
An extract of Lentinus edodes mycelium used herein is preferably obtained by, but not specifically limited to, the following process.
Lentinus edodE~s spawn is inoculated on a solid medium based on bagasse (sugar cane residue) and defatted rice bran to grow mycelia, and then the solid medium containing the grown mycelia is delignified to enable about 30~ by weight or less to pass through a 12-mesh sieve.
To this delign:ified solid medium are added water and one or more enzymes selected from cellulase, protease or glucosidase while maintaining said solid medium at a temperature of around 30-55°C, and said solid medium is crushed and ground in the presence of said enzyme so that at least '70~ by weight of bagasse fiber is able to pass through a 12-mesh sieve. Then, the temperature is raised to 80-100"C to ensure inactivation of the enzyme and sterilization, and the resulting suspension is filtered to give an extract of Lentinus edodes mycelium.
The extract of Lentinus edodes mycelium as prepared above may be directly used in LAK activity enhancers or LAK
activity-enhancing farmulations of the present invention, but conveniently concentrated and freeze-dried into powder to be stored and used in various forms. The freeze-dried product is a brown powder with hygroscopic characteristics and has a peculiar taste and odor.
LAK activity e:nhancers containing the prepared extract of Lentinus edodes mycelium (or the extract of Lentinus edodes mycE:lium on its own) may be used in LAK
therapy in place of IL-2 to enhance the immune activity against tumor of thE: subject .
LAK therapy gE:nerally comprises the steps of culturing in vitro :Lymphocytes from the subject with IL-2 to induce LAK cells from the lymphocytes, and reinfusing the LAK cells into the subject. At the step of reinfusing LAK cells into thE: subject, IL-2 may be (directly) cocurrent:Ly administered.
LAK activity enhancers cantaining the extract of Lentinus edodes mycelium of the present invention may be cultured :in vitro with lymphocytes from the subject (human, animals, etc.) to :induce LAK cells or may be (directly) cocurrently administered to the subject at the step of reinfusing the cu:Ltu.red lymphocytes to the subject. They may be used in combination with other antitumor and/or anticancer agents.
LAK activity enhancers containing the extract of Lentinus edodes mycelium of the present invention may be directly added to peripheral blood collected from the subject, but preferably sterilized with acetone before being added.
LAK activity e:nhancers containing the extract of Lentinus edodes mycE:lium of the present invention can be directly added to pE:ripheral blood collected from the subject preferably at a concentration expressed as the extract o~f Lentinus edodes mycelium of 1 ~g - 1 mg/106 lymphocytes, more preferably 10 ~g - 100 ~g/106 lymphocytes, most preferably 10 Ecg - 50 ~g/106 lymphocytes. The dose per ml of periphera:L blood, which partially depends on the level of lymphocytes in peripheral blood, approximately represents 1 ~g - 2.5 mg, preferably 10 ~.g - 250 dug, more preferab7_y 10 ~.g - 125 ~.g.
LAK activity-~e:nhancing formulations comprising an LAK
activity enhancer containing the extract of Lentinus edodes mycelium of the present invention can be directly administered to the subject to enhance the anti-tumor immune activity oi_' the subject, thereby providing an effect comparable to the effect elicited by LAK therapy using IL-2.
Induction of L.AK cells in the subject such as a human or animals by directly administering the LAK activity enhancers of the present invention may make it possible to reduce physical stress associated with the isolation of a great number of leukocytes and the high cost resulting from producing mass culture. In addition, the use of LAK
activity-enhancers o~f the present invention can also reduce the risk of contamination during the isolation of blood from the subject and. reinfusion can also be reduced.
LAK activity-enhancing formulations of the present invention are administered most preferably, but are not limited to, the oral. route in view of the least stress imposed on the su~bje:ct and the like. They may also be administered via intravenous, intracutaneous, subcutaneous or intramuscular injjection, or percutaneous, nasal, enteral or other routes.
Suitable dosage forms of LAK activity-enhancing formulations of the present invention include, but are not limited to, tablets, capsules, powders, granules, solutions, syrups, injections, creams, ointments, patches, sprays, suppositories, etc.
Pharmaceutically acceptable carriers that can be optionally mixed witlh LAK activity-enhancing formulations of the present invention include, but not limited to, excipients such as lactose, dextrose, starch, crystalline cellulose; binders such as starch, gelatin, methyl cellulose" polyvinylpyrrolidone; disintegrators such as starch, c<~lcium carboxymethylcellulose, carboxymethyl starch; lubricants such as talc, stearates; coating agents such as sucrose, t=alc, gelatin; as well as various brighteners, flavoring agents, colorants, corrigents, solubilizers, stabilizers, suspending agents, absorbefacients or the like known in the art depending on the purpose. For use as injections, various diluents commonly used in this field of art such as water or ethyl alcohol can be used.
The route oi: administration, dose, frequency or others of LAK act:ivi.ty-enhancing formulations of the present invention are determined taking into account the age, weight, condition and other factors of the subject.
The extract of Lentinus edodes mycelium contained in LAK activity-enhancing formulations of the present invention is highly safe as has been traditionally ingested as a component of food. Therefore, the dose is not be strictly limited. For example, the extract of Lentinus edodes mycelium is normally administered preferably at a dose of 100 mg - 10000 mg 2-3 times daily, more preferably 500 mg - 5000 mg three times daily, most preferably 1000 mg - 1500 mg three times daily. It may be administered in combination with other drugs such as antitumor and/or anticancer. agents such as immunopotentiators, antibiotics, analgesics , antiinf l~ammatory agents , etc .
LAK activity-enhancing formulations of the present invention can also be provided in the form of a food.
Preferred forms of food include powders, granules, pastes, jellies, Eac. Granules desirably are supplemented with sugars such as lactose to add a sweet taste. LAK activity-enhancing formulations of the present invention can also be provided _Ln the form of a drink. These foods or drinks may be supplernented with vitamins, minerals such as calcium, alcohols, deodorants such as polyphenols in addition to the extract of Lentinus edodes mycelium. These foods or drinks may include the categories of specific health foods, medical foods or the like.
LAK activity-enhancing formulations of the present invention can also b~e provided in the form of a feed or feed additive. LAK activity-enhancing formulations of the present invention cam be used as a feed or feed additive for domestic animals; to treat and/or prevent tumor occurring in the domestic animals or to treat and/or prevent bacterial ox' viral infections in domestic animals.
As a result, the amount of therapeutic agents such as antibiotics currently used can be reduced, thereby reducing farming costs. Another advantage is that the period during which shipment of animals is suspended due to the administration of antibiotics can be shortened.
LAK activity enhancement with LAK activity enhancers of the present invention can be confirmed by, but not limited to, the pracedure below according to the method of Takagi et al. (Cli.nical Immunology, 19:245-249, 1987).
T~AK activity tes LAK activity can be determined by S~Cr release assay, [3HJ uridine assay or the like. In terms of convenience and objectivity, the SlCr release assay is preferably used.
The SlCr releasE; assay is one of methods for in vitro determining the cytotoxicity against target cells of LAK
cells induced from lymphocytes treated with an LAK activity enhancer, and comprises the steps of:
(i) adding SlCr-labeled sodium chromate to the target cells to label th<: target cells, (ii) reacting the labeled target cells with induced LAK cells, and (ii:i) measuring the amount of SlCr released into the cell culture supernatant from the target cells bursted by the LAK cells.
Subcultured cells used as target cells in the 5lCr release assay are preferably Daudi cells or Raji cells.
Target cells culturE:d in a culture flask or the like are collected by centric=ugation, and labeled with 5lCr via incubation in a 5~ (:OZ incubator at 37°C for 1-2 hours after 100-150 ~Ci 5lCr labeled-sodium chromate has been added. Culture media for target cells are those suitable for the growth of the cells used, and include, for example, liquid media such a;s RPMI 1640 or F-12 appropriately supplemented with serum, antibiotics, etc.
Cultured target cells are washed with PBS three times, and then suspended in a culture medium at a density of 1 x 106 cells/ml to serv<, for assay.
To induce LAK cells, lymphocytes are isolated from peripheral blood of the subject. Heparin is added to peripheral blood from the subject, and monocytes at the interface are separated by density gradient centrifugation on Ficoll~-Conray solution (s. g. - 1.077). The separated monocytes are washed with PBS (pH 7.4, without Ca and Mg) 2-3 times and then suspended in a culture medium (preferab:ly, RPMI :1640 medium (Gibco) containing FBS
(inactivated fetal bovine serum) and/or antibiotics, if desired) at a density of 1 x 106 cells /ml. This suspension is transferred to a Petri dish which has been precoated with autoserum (plasma) at 37°C for 15 minutes, and incubated at :37°C for 1 hour. Unattached cells are recovered as lymphocyte fractions.
The lymphocytes as prepared above are suspended in a culture medium at a predetermined concentration, preferably 1 x 105-106 cells/ml.
The extract of Lentinus edodes mycelium is added to the medium as used f:or preparing the lymphocyte-suspending solution at a final concentration of about 1-100 ~.g/ml.
Predetermined amounts of the lymphocyte-suspending solution and extract:-containing medium are added to wells of a culture plate. In parallel, predetermined amounts of the lymphocyte-floai~ing solution and the medium not containing the extract of Lentinus edodes mycelium are added to wells as an extract-free control. Then, the culture p7_ate is statically incubated in a COz incubator at room temperature for 3 days.
When a 96-we'll U-bottom microtest plate is used, for example, 100 ~1 of t:he lymphocyte-floating solution is preferably added t:o each well at a density of 1 x 10' to 1 x 105 cells/well. The number of cells per well can be appropriately determined by those skilled in the art in accordance with the well volume, the activity of LAK cells, the sensitivity of target cells to LAK cells, etc.
Thus, lymphocytes derived from the subject are cultured :in the presence of the extract of Lentinus edodes mycelium of the present invention at various concentrations (including zero) to induce effector cells. As used herein, the term effector cells refers to cells subjected to said culture treatment far 3 days and includes both lymphocytes cocultured with an L~AK activity enhancer (lymphocytes treated with an LAK activity enhancer) and lymphocytes cultured in the medium alone without LAK activity enhancer (lymphocytes treated under the extract-free condition).
Cultured lymphocytes (effector cells) are recovered from the well and rE:suspended in fresh RPMI 1640 medium containing 10~ FBS ( 1 x 106 cel7_s /ml ) .
In the assay for determining cytotoxicity, each well containing said target cells is further supplied with a predetermined amouni~ of the floating solution containing said various effector cells (lymphocytes treated with LAK
active enhancer or those under the extract-free condition) at a density of 1 x 105 - 1 x 106 cells/ml for experimental dissociation. 1N HC1 in an amount equal to-the effector-floating solution for maximum dissociation, or the medium alone in an amount equal to the effector-floating solution S for natural dissociation. Then, cells are collected at the bottom of the well of the cultuze plate using a plate centrifuge or the like, and then incubated in a S% CO
incubator at 3'7'C fo:r a predetermined period (for example, 2-5 hours:l.
After completion of the incubation, the radioactivity of S~Cr released in i:he culture supernatant in the culture supernatant in each well is measured using a scintillation counter or the like_ The term "maximum dissociation" refers to the total amount of S'Cr relea;:ed when all the target cells are destroyed. Maximum dissociation can be determined by, for example, lysing targret cells via incubation in a culture medium containing 1N HC1 under predetermined conditions in the absence of effector cells and measuring the amount of SlCr released in the culture supernatant.
The term "experimental dissociation" refers to the amount of S=Cr releaaed by cytotoxicity of effector cells against target cells and natural death of target cells when effector cells are added to target cells. Experimental dissociation can be determined by, for example, incubating target cells under the same conditions as those for maximum dissociation with the exception of adding effector cells in place of 1N HC1 to the culture medium and measuring the amount of SlCr released in the culture supernatant.
The term "natural dissociation" refers to the amount of SlCr released by natural death of target cells. Natural dissociation can be determined by, for example, incubating target cells under the same conditions as for maximum dissociation with exception of containing neither 1N HCl nor effector cells and measuring the amount of SlCr released :in the culture supernatant.
The activation level of LAK cells is evaluated on the basis of 'the LAK activity calculated by equation (1) below.
Experimental dissociation(cpm)-Natural dissociation(cpm) LAK activity _ ---- x 100 Maximum dissociation(cpm)-Natural dissociation(cpm) Equation (1) The disclosure of JPA Nos. 353927/98 and 353928/98 on which the present application is based to claim the priority are incorporated herein as a whole as reference.
The following examples further illustrate the present invention but should. not be taken as limiting the scope of the invention thereto. It will be appreciated by those skilled in the art that various changes and modifications can be made without departing from the spirit of the present invention.
E3~AMPLES
A solid med:ium~ consisting of 90 parts by weight of bagasse and 10 parts by weight of rice bran was soaked with an appropriate amount of pure water, and then inoculated with Lentinus edodes spawn and allowed to stand in an incubator at controlled temperature and humidity to grow mycelia. After mycelia spread over the solid medium, the bagasse base was delignified to enable 24~ by weight or less eto pass through a 12-mesh sieve. To 1.0 kg of this delignified medium were added 3.5 L of pure water and 2.0 g of purified cellulase while maintaining the solid medium at 40°C to prepare a medium-containing mixture.
Then, the medium-containing mixture was circulated by a variable speed gear pump, during which the solid medium was crushed and ground at the gears for about 200 minutes so that about 80~ by weight of bagasse fiber is able to pass through a 12~-me,sh sieve. The medium-containing mixture was crushed and ground while the temperature of said mixture was gradually increased. Then, the medium-containing mixture was further heated to 90°C to ensure inactivation of the enzyme and sterilization and allowed to stand at 90°C for :30 minutes. The resulting medium-containing mixture was filtered through a 60-mesh filter cloth to give an extract of Lentinus edodes mycelium solution, which was concentrated and then converted into a freeze-dried powder.
The extract of Lentinus edodes mycelium as prepared above contained 25.:3 (w/w) carbohydrates determined by the phenol-sulfuric acid method, 19.7 (w/w) proteins determined by the Lowry method and 2.6~ (w/w) polyphenols determined by the F'olin-Denis method using gallic acid as standard. The extract of Lentinus edodes mycelium further contains 8~ crude fat, 22~ crude ash and about 20~ soluble nitrogen-free matE:rials other than carbohydrates.
The extract of Lentinus edodes mycelium had a sugar composition (~) as follows: Xyl 15.2, Ara 8.2, Man 8.4, Gul 39.4, Gal 5.4, GlcN 12.0, GluUA 11.3.
Exam In a 2~ Comparison of LAK activities of the extract of r.Pnt; n_ms edodes myce~.? um and IL-2 The extract of Lentinus edodes mycelium obtained in Example 1 was directly administred to peripheral blood collected from the subject to compare the LAK activity-enhancing effect with that of IL-2.
Heparin was added to peripheral blood from subject A, and monocytes at the:.interface were separated by density gradient centrifugation on Ficoll-Conray solution (s.g. -1.077). The separated monocytes were washed with PBS (pH
7.4, without Ca and Mg) twice and then suspended in RPMI
1640 medium (Gibco) containing 10~ FBS (inactivated fetal bovine serum) at a density of 1 x 106 cells /ml. This cell suspension was transferred to a Petri dish which had been precoated with auto.>erum (plasma) at 37°C for 5 minutes followed by incubation at 37°C for 1 hour, and then unattached cells were recovered as lymphocyte fractions.
The lymphocytes as prepared above were suspended in RPMI 1640 medium (Gibco) containing 10~ FBS (inactivated serum) at 1 x 106 ~:ells /ml.
LAK activity enhancer-containing solutions were prepared by adding IL-2 at a final concentration of 25 U/ml or the extract of Lentinus edodes mycelium obtained in Example 1 at fina7_ concentrations of 1, 10, 100 or 1000 ~,g/ml to RPMI 164C1 medium ( Gibco ) containing 10~ FBS
(inactivated serum).
To each well. o:f a 96-well U-bottom microtest plate were added 100 ~l of the lymphocyte-suspending solution and 100 ~l of IL-2 or the LAK activity enhancer-containing solution containing the extract of Lentinus edodes mycelium at varioua concentrations (a total of 20U ~1). As an extract-free control, 100 ~1 of the lymphocyte-suspending solution and 100 ~w,1 of the extract-free medium (RPMI 1640 medium (G:ibco) containing 10~ FBS (inactivated serum)) were added. Then, the culture plate was statically incubated in a COZ incubator at room temperature for 3 days.
Cultured lymphocytes (effector cells) were recovered from the well and resuspended in fresh RPMI 1640 medium containing 10~ FBS a.t a concentration of 1 x 106 cells/ml.
Target cells (Daudi cells from Dainippon Pharmaceutical) subc:ultured in RPMI 1640 medium containing 10~ FBS were recovered by centrifugation, and incubated with 100-150 Er,Ci/ 106 cells of SICr labeled-sodium chromate (New England Nuclear) in a 5~ COZ incubator at 37°C for 1 hour. Cultured cells labeled with SlCr were washed with PBS three times, and then suspended in RPMI 1640 medium containing loo FBS at a concentration of 1 x 106 cells/ml.
A 50 ~1 aliquot (5 x 10' cells/well) of labeled target cells was added to each of wells of the microtest plate for maximum dissociation, natural dissociation and experimental dissociation. In addition, 100 ul of 1N HC1 was added 'to each well for maximum dissociation, 100 t-il of RPMI 1640 medium cont.ainirig 10% FBS was added to each well for natural dissoc:iat.i_on, and 100 ul of effector cells treated with 10 ~glml of the extract of Lentinus edodes mycelium of the present invention at concentrations of 1, 10, 100 and 1000 Ng/mal or 25 U/ml of IL-2 or the medium alone were added to each well for experimental. dissociation.
Here, each well for experimental dissociation, maximum dissociation and natural dissociation was not located around the outer periphery of the plate.
Then, the microtest plate was centrifuged at 800 rpm for 5 minutes on a plate centrifuge to collect cells at the bottom of the well, a.nd then incubated in a 5% CO=
incubator at 37aC f.or 3.5 hours_ The culture supernatant in each well was collected by SOKEN-PET ~-96 from t:he incubated plate, and the radioactivity was measured in a Y-scintillation counter.
LAK activity was calculated by equation (1) above.
The results are shown in Table 1 and Fig. 1_ Table 1 Test No. Inducer LAK activity 1 None 11~
2 IL-2 (final concentration: 25 U/ml) 55~
3 Extract of Lentinus edodes mycelium 14~
(ffinal concE;ntration: 1 ~g/ml) 4 Extract of I~entinus edodes mycelium 20~
( final concE;ntration : 10 ~g/ml ) Extract of Lentinus edodes mycelium 14~
(final concE:ntration: 100 ~g/ml) In order to examine the toxicity of the extract of Lentinus edodes mycelium to lymphocytes, lymphocytes was 5 treated with the extract of Lentinus edodes mycelium as prepared in Example 1 at a final concentration of 1 mg/ml by the same procedure as described above and observed under a microscope to show that almost 100 of lymphocytes survived (data not shown).
Example 3 : O~timal.~~~~~~~+rat; nn of the extract of Lentinus edodes mvcelium f~i;AK activitv The optimal concentration of the extract of Lentinus edodes mycelium to be directly added to peripheral blood collected from the subject was examined. LAK activity was calculated in the same manner as described in Example 2 with exception that the extract of Lentinus edodes mycelium as prepared in Example 1 was added at final concentrations of 1, 5, 10 and 100 ~g/ml to peripheral blood collected from subjects B and C.
The results are shown in Table 2 and Fig. 2.
As shown in Table 2 and Fig. 2, the extract of Lentinus E:dodes mycelium of the present invention shows the highest LAK activity when it is added at a final concentration of 10 ~g/ml or 50 ~.g/ml (i.e. 10 ~g or 50 ~,g per 106 lymphocytes).. The dose to peripheral blood also depends on the proportion of lymphocytes in peripheral blood of the subjE:ct, but approximately corresponds to about 10-25 ~g or 50-125 ~.g of the extract of Lentinus edodes mycelium pE:r 1 ml of peripheral blood.
Table 2 Test No. Inducer LAK activity Subject B C
1 None 13~ 27~
2 Extract of Lentinus edodes mycelium 18~ 28~
(ffinal concentration: 1 ~.g/ml) 3 Extract of Lentinus edodes mycelium 19~ 29~
(f_inal concentration: 5 ~g/ml) 4 Extract of Lentinus edodes mycelium 21~ 34~
( f:inal concentration : 10 ~g/ml ) 5 Extract of Lentinus edodes mycelium 19~ 36~
(f:inal concentration: 50 ~g/ml) 6 Extract of Lentinus edodes mycelium 15~ 30~
(f.inal concentration: 100 ~.g/ml) LAK activity-enhancing effect of an immunopotentiator Krestin was examined to compare it with the effect of the extract of Lentinus edodes mycelium of the present invention. LAK activity-enhancing effect of Krestin was evaluated in the Name manner as described in Example 2 with exception that Krestin (from Kureha Chemical Industry) was added to peripheral blood collected from subject D at a final concentration of 1 mg/ml.
The final concentration of 1 mg/ml is the concentration said to be the most effective when Krestin is used to induce LAK cells.
The results are shown in Table 3 and Fig. 3.
As shown in Table 3 and Fig. 3, the LAK-enhancing activity ~of Krest:in was lower than that of a control not containing LAK activity enhancer even at the optimal concentration of 1 m~g/ml.
Table 3 Test No. Inducer LAK activity 1 None 14~
2 Krestin (final concentration: 1 mg/ml) 13~
Examples 4: Determin~~tion of LAK activity before and after administration of_~tormu~at~on containing the extract of An LAK activity-enhancing formulation containing the extract of Lentinus edodes mycelium prepared in the same manner as described in Example 1 was administered to subjects B, C, E and F to evaluate the LAK activity-enhancing effect.
This LAK activity-enhancing formulation comprises 0.6 g of the extract of. Lentinus edodes mycelium and 2.4 g of lactose per 3 g of the formulation, and was administered at a dose of 6 g (1.2 g as the extract of Lentinus edodes mycelium) three t:Lmes daily or a total of 3.6 g/day expressed as the extract of Lentinus edodes mycelium for a week. Peripheral blood was collected from each subject before administration and about 2 hours after the final administration, and LAK activity was determined in the same manner as described in Example 2.
The results are shown in Table 4 and Fig. 4.
As shown in 'ra.ble 4 and Fig. 4, the extract of Lentinus edodes mycelium of the present invention remarkably increasect LAK activity in all of subjects B, C, E and F.
Table 4 r.,~K act~v~tv Subject B C E F
Before administration of LEM 13~ 27~ 14~ 18~
After administration of LEM 40~ 43~ 18~ 28~
In order to examine side effects caused by the administration of the extract of Lentinus edodes mycelium, biochemical blood tests were performed on subjects B, C and E. The results are shown in Table 5. Throughout the period for administration of the extract of Lentinus edodes mycelium, subjects B, C, E and F did not show any side effects, such as generalized fatigue, chills, fever or digestive system symptoms.
As also shown in Table 5, any side effects were not observed by administration of the extract of Lentinus edodes mycelium of the present invention.
Table 5 Subject B ~
Subject C Subject E
__ Administration for 1 week __ After Before After Before After Be f or e _ _ 5.94 5.85 5.08 8.01 7.47 WBC (x10'/wl) _ _ 5.45 RBC (x10'/wl) 4.5f~ 4.61 4.99 4.85 4.89 4.65 Hb (g/dl) 14.~: 14.2 14.4 13.8 15.5 14.8 Ht (8) 42.~: 42.8 43.6 42.1 44.8 42.9 MCV (fl) 92.5 92.8 87.4 86.8 91.6 92.3 MCH (pg) 3:1..7.30.8 28.9 28.5 31.7 31.8 MCHC (g/dl) 33.fi 33.2 33.0 32.8 34.6 34.5 PLT (x10/~1) 1!a.l 18.5 23.9 19.3 29.2 29.7 RDW-SD (fl) 4::1. 44.6 93.5 43.6 43.7 44.8 ;' RDW-CV (~) 12. 13.0 13.4 13.6 13.0 13.3 ES
PDW (fl) 14.0 13.4 11.6 12.6 13.3 13.2 MPV (fl) 1.1.1 10.9 10.0 10.2 10.6 10.4 P-LCR (~) 34.7. 32.5 24.9 27.6 30.1 28.8 Stab 0.5 0.5 1.0 0.0 0.5 0.0 Seg 54.0 48.5 56.0 43.5 46.5 38.5 L~ 3"?.5 40.5 38.0 43.5 28.5 35.0 Mono 9.5 7.5 3.0 9.5 i 11.5 8.5 Eo 2.5 3.0 1.5 1.0 ' 12.0 15.5 Baso 1.0 0.0 0.5 0.5 0.5 0.5 (g/dl) E..6 6.9- 7.5 8.0 7.7 T P ~ 7.7 ALB (g/dl) =.9 4.1 ~ 4.3 4.8 4.7 4.4 A/G 1.44 1.46 1..34 1.50 1.57 1.33 TTT (SHU) 3.9 5.0 8.D B.1 5.3 3.0 ZTT (Kunk) 8.6 8.4 12.6 12.4 6.3 4.9 U-N (mg/dl) 10.4 10.3 11.2 11.8 11.6 10.2 U-A (mg/dl) 5.6 5.9 5.4 5.2 6.1 5.9 Creatinine (mg/dl)0.67 0.71 0.82 0.67 0.72 0.84 Overall BIL (mg/dl)0.6 0.7 0.5 0.6 0.5 0.5 Direct BIL (mg/dl)0.1 0.1 0.0 0.0 0.1 0.1 !
Indirect BIL 0.5 0.6 0.5 0.6 0.4 0.4 (mg/dl) ~
GLU (mg/dl) :124 104 94 111 115 114 AST (GOT) (Iil/1)15 13 18 16 33 33 ALT (GPT) (ILf/1)29 30 20 20 45 57 LD (LDH) (IU%1) 142 139 171 152 380 343 CK (CPK) (IU/1) :L21 131 227 177 317 224 ALP (IU/1) I :1.31136 156 148 174 162 y-GT (IU/1) 12 14 23 23 225 240 LAP (IU/1) 45 40 80 73 134 120 CHE (IU/1) 2358 2593 2749 3191 3299 , 2779 AMY (IU/1) 78 87 ~ 93 59 54 T-CHO (mg/dlj x:08 232 ! 200 255 243 HDLCHO (mg/d:) 50 50 44 40 47 47 T-G (mg/dl) ~ 1.35 210 270 303 451 359 ', Na (mEq/1) 147. 142 140 141 139 141 K (mEq/1) 3.7 3.9 4.1 3.8 4.0 4.2 Cl (mEq/1) lOEo 107 105 107 103 104 Ca (mEq/1) 4.~1 4.4 4.6 4.4 4.9 4.7 I-P (mg/dl) :3.:5 3.1 3.0 2.7 3.5 3.2 RA test (-.! (-) (-) (-) (-) (-) CRP (mg/dl) 0.03 0.06 0.01 0.07 0.12 ~ 0.02 ANA <40 <40 I <40 <40 <40 <40 ANA pattern (-1 (-) (-) (-) (-) (-) CH50 (CH50/m.l) ;53.0 33.0 42.0 53.0 52.0 45.0 LAK enhancers containing the extract of Lentinus edodes mycelium of t:he present invention showed similar effects to those of IL-2 with no direct toxicity to lymphocytes when i:hey were directly administered to peripheral blood. They are available at far lower cost as compared with IL-2. Therefore, they can be substituted for expensive IL-2 which also has the disadvantage of causing strong side effects.
Formulations containing the extract of Lentinus edodes mycelium of the present invention could remarkably increase :LAK activity with no side effects even by direct administration. 'rhe:refore, formulations containing the extract of Lentinus edodes mycelium of the present invention are useful- as an immunopotentiator capable of increasing LAK activity with a benign side-effect profile by direct administration such as oral administration without using LAK 'therapy.
1640 medium (Gibco) containing 10~ FBS (inactivated fetal bovine serum) at a density of 1 x 106 cells /ml. This cell suspension was transferred to a Petri dish which had been precoated with auto.>erum (plasma) at 37°C for 5 minutes followed by incubation at 37°C for 1 hour, and then unattached cells were recovered as lymphocyte fractions.
The lymphocytes as prepared above were suspended in RPMI 1640 medium (Gibco) containing 10~ FBS (inactivated serum) at 1 x 106 ~:ells /ml.
LAK activity enhancer-containing solutions were prepared by adding IL-2 at a final concentration of 25 U/ml or the extract of Lentinus edodes mycelium obtained in Example 1 at fina7_ concentrations of 1, 10, 100 or 1000 ~,g/ml to RPMI 164C1 medium ( Gibco ) containing 10~ FBS
(inactivated serum).
To each well. o:f a 96-well U-bottom microtest plate were added 100 ~l of the lymphocyte-suspending solution and 100 ~l of IL-2 or the LAK activity enhancer-containing solution containing the extract of Lentinus edodes mycelium at varioua concentrations (a total of 20U ~1). As an extract-free control, 100 ~1 of the lymphocyte-suspending solution and 100 ~w,1 of the extract-free medium (RPMI 1640 medium (G:ibco) containing 10~ FBS (inactivated serum)) were added. Then, the culture plate was statically incubated in a COZ incubator at room temperature for 3 days.
Cultured lymphocytes (effector cells) were recovered from the well and resuspended in fresh RPMI 1640 medium containing 10~ FBS a.t a concentration of 1 x 106 cells/ml.
Target cells (Daudi cells from Dainippon Pharmaceutical) subc:ultured in RPMI 1640 medium containing 10~ FBS were recovered by centrifugation, and incubated with 100-150 Er,Ci/ 106 cells of SICr labeled-sodium chromate (New England Nuclear) in a 5~ COZ incubator at 37°C for 1 hour. Cultured cells labeled with SlCr were washed with PBS three times, and then suspended in RPMI 1640 medium containing loo FBS at a concentration of 1 x 106 cells/ml.
A 50 ~1 aliquot (5 x 10' cells/well) of labeled target cells was added to each of wells of the microtest plate for maximum dissociation, natural dissociation and experimental dissociation. In addition, 100 ul of 1N HC1 was added 'to each well for maximum dissociation, 100 t-il of RPMI 1640 medium cont.ainirig 10% FBS was added to each well for natural dissoc:iat.i_on, and 100 ul of effector cells treated with 10 ~glml of the extract of Lentinus edodes mycelium of the present invention at concentrations of 1, 10, 100 and 1000 Ng/mal or 25 U/ml of IL-2 or the medium alone were added to each well for experimental. dissociation.
Here, each well for experimental dissociation, maximum dissociation and natural dissociation was not located around the outer periphery of the plate.
Then, the microtest plate was centrifuged at 800 rpm for 5 minutes on a plate centrifuge to collect cells at the bottom of the well, a.nd then incubated in a 5% CO=
incubator at 37aC f.or 3.5 hours_ The culture supernatant in each well was collected by SOKEN-PET ~-96 from t:he incubated plate, and the radioactivity was measured in a Y-scintillation counter.
LAK activity was calculated by equation (1) above.
The results are shown in Table 1 and Fig. 1_ Table 1 Test No. Inducer LAK activity 1 None 11~
2 IL-2 (final concentration: 25 U/ml) 55~
3 Extract of Lentinus edodes mycelium 14~
(ffinal concE;ntration: 1 ~g/ml) 4 Extract of I~entinus edodes mycelium 20~
( final concE;ntration : 10 ~g/ml ) Extract of Lentinus edodes mycelium 14~
(final concE:ntration: 100 ~g/ml) In order to examine the toxicity of the extract of Lentinus edodes mycelium to lymphocytes, lymphocytes was 5 treated with the extract of Lentinus edodes mycelium as prepared in Example 1 at a final concentration of 1 mg/ml by the same procedure as described above and observed under a microscope to show that almost 100 of lymphocytes survived (data not shown).
Example 3 : O~timal.~~~~~~~+rat; nn of the extract of Lentinus edodes mvcelium f~i;AK activitv The optimal concentration of the extract of Lentinus edodes mycelium to be directly added to peripheral blood collected from the subject was examined. LAK activity was calculated in the same manner as described in Example 2 with exception that the extract of Lentinus edodes mycelium as prepared in Example 1 was added at final concentrations of 1, 5, 10 and 100 ~g/ml to peripheral blood collected from subjects B and C.
The results are shown in Table 2 and Fig. 2.
As shown in Table 2 and Fig. 2, the extract of Lentinus E:dodes mycelium of the present invention shows the highest LAK activity when it is added at a final concentration of 10 ~g/ml or 50 ~.g/ml (i.e. 10 ~g or 50 ~,g per 106 lymphocytes).. The dose to peripheral blood also depends on the proportion of lymphocytes in peripheral blood of the subjE:ct, but approximately corresponds to about 10-25 ~g or 50-125 ~.g of the extract of Lentinus edodes mycelium pE:r 1 ml of peripheral blood.
Table 2 Test No. Inducer LAK activity Subject B C
1 None 13~ 27~
2 Extract of Lentinus edodes mycelium 18~ 28~
(ffinal concentration: 1 ~.g/ml) 3 Extract of Lentinus edodes mycelium 19~ 29~
(f_inal concentration: 5 ~g/ml) 4 Extract of Lentinus edodes mycelium 21~ 34~
( f:inal concentration : 10 ~g/ml ) 5 Extract of Lentinus edodes mycelium 19~ 36~
(f:inal concentration: 50 ~g/ml) 6 Extract of Lentinus edodes mycelium 15~ 30~
(f.inal concentration: 100 ~.g/ml) LAK activity-enhancing effect of an immunopotentiator Krestin was examined to compare it with the effect of the extract of Lentinus edodes mycelium of the present invention. LAK activity-enhancing effect of Krestin was evaluated in the Name manner as described in Example 2 with exception that Krestin (from Kureha Chemical Industry) was added to peripheral blood collected from subject D at a final concentration of 1 mg/ml.
The final concentration of 1 mg/ml is the concentration said to be the most effective when Krestin is used to induce LAK cells.
The results are shown in Table 3 and Fig. 3.
As shown in Table 3 and Fig. 3, the LAK-enhancing activity ~of Krest:in was lower than that of a control not containing LAK activity enhancer even at the optimal concentration of 1 m~g/ml.
Table 3 Test No. Inducer LAK activity 1 None 14~
2 Krestin (final concentration: 1 mg/ml) 13~
Examples 4: Determin~~tion of LAK activity before and after administration of_~tormu~at~on containing the extract of An LAK activity-enhancing formulation containing the extract of Lentinus edodes mycelium prepared in the same manner as described in Example 1 was administered to subjects B, C, E and F to evaluate the LAK activity-enhancing effect.
This LAK activity-enhancing formulation comprises 0.6 g of the extract of. Lentinus edodes mycelium and 2.4 g of lactose per 3 g of the formulation, and was administered at a dose of 6 g (1.2 g as the extract of Lentinus edodes mycelium) three t:Lmes daily or a total of 3.6 g/day expressed as the extract of Lentinus edodes mycelium for a week. Peripheral blood was collected from each subject before administration and about 2 hours after the final administration, and LAK activity was determined in the same manner as described in Example 2.
The results are shown in Table 4 and Fig. 4.
As shown in 'ra.ble 4 and Fig. 4, the extract of Lentinus edodes mycelium of the present invention remarkably increasect LAK activity in all of subjects B, C, E and F.
Table 4 r.,~K act~v~tv Subject B C E F
Before administration of LEM 13~ 27~ 14~ 18~
After administration of LEM 40~ 43~ 18~ 28~
In order to examine side effects caused by the administration of the extract of Lentinus edodes mycelium, biochemical blood tests were performed on subjects B, C and E. The results are shown in Table 5. Throughout the period for administration of the extract of Lentinus edodes mycelium, subjects B, C, E and F did not show any side effects, such as generalized fatigue, chills, fever or digestive system symptoms.
As also shown in Table 5, any side effects were not observed by administration of the extract of Lentinus edodes mycelium of the present invention.
Table 5 Subject B ~
Subject C Subject E
__ Administration for 1 week __ After Before After Before After Be f or e _ _ 5.94 5.85 5.08 8.01 7.47 WBC (x10'/wl) _ _ 5.45 RBC (x10'/wl) 4.5f~ 4.61 4.99 4.85 4.89 4.65 Hb (g/dl) 14.~: 14.2 14.4 13.8 15.5 14.8 Ht (8) 42.~: 42.8 43.6 42.1 44.8 42.9 MCV (fl) 92.5 92.8 87.4 86.8 91.6 92.3 MCH (pg) 3:1..7.30.8 28.9 28.5 31.7 31.8 MCHC (g/dl) 33.fi 33.2 33.0 32.8 34.6 34.5 PLT (x10/~1) 1!a.l 18.5 23.9 19.3 29.2 29.7 RDW-SD (fl) 4::1. 44.6 93.5 43.6 43.7 44.8 ;' RDW-CV (~) 12. 13.0 13.4 13.6 13.0 13.3 ES
PDW (fl) 14.0 13.4 11.6 12.6 13.3 13.2 MPV (fl) 1.1.1 10.9 10.0 10.2 10.6 10.4 P-LCR (~) 34.7. 32.5 24.9 27.6 30.1 28.8 Stab 0.5 0.5 1.0 0.0 0.5 0.0 Seg 54.0 48.5 56.0 43.5 46.5 38.5 L~ 3"?.5 40.5 38.0 43.5 28.5 35.0 Mono 9.5 7.5 3.0 9.5 i 11.5 8.5 Eo 2.5 3.0 1.5 1.0 ' 12.0 15.5 Baso 1.0 0.0 0.5 0.5 0.5 0.5 (g/dl) E..6 6.9- 7.5 8.0 7.7 T P ~ 7.7 ALB (g/dl) =.9 4.1 ~ 4.3 4.8 4.7 4.4 A/G 1.44 1.46 1..34 1.50 1.57 1.33 TTT (SHU) 3.9 5.0 8.D B.1 5.3 3.0 ZTT (Kunk) 8.6 8.4 12.6 12.4 6.3 4.9 U-N (mg/dl) 10.4 10.3 11.2 11.8 11.6 10.2 U-A (mg/dl) 5.6 5.9 5.4 5.2 6.1 5.9 Creatinine (mg/dl)0.67 0.71 0.82 0.67 0.72 0.84 Overall BIL (mg/dl)0.6 0.7 0.5 0.6 0.5 0.5 Direct BIL (mg/dl)0.1 0.1 0.0 0.0 0.1 0.1 !
Indirect BIL 0.5 0.6 0.5 0.6 0.4 0.4 (mg/dl) ~
GLU (mg/dl) :124 104 94 111 115 114 AST (GOT) (Iil/1)15 13 18 16 33 33 ALT (GPT) (ILf/1)29 30 20 20 45 57 LD (LDH) (IU%1) 142 139 171 152 380 343 CK (CPK) (IU/1) :L21 131 227 177 317 224 ALP (IU/1) I :1.31136 156 148 174 162 y-GT (IU/1) 12 14 23 23 225 240 LAP (IU/1) 45 40 80 73 134 120 CHE (IU/1) 2358 2593 2749 3191 3299 , 2779 AMY (IU/1) 78 87 ~ 93 59 54 T-CHO (mg/dlj x:08 232 ! 200 255 243 HDLCHO (mg/d:) 50 50 44 40 47 47 T-G (mg/dl) ~ 1.35 210 270 303 451 359 ', Na (mEq/1) 147. 142 140 141 139 141 K (mEq/1) 3.7 3.9 4.1 3.8 4.0 4.2 Cl (mEq/1) lOEo 107 105 107 103 104 Ca (mEq/1) 4.~1 4.4 4.6 4.4 4.9 4.7 I-P (mg/dl) :3.:5 3.1 3.0 2.7 3.5 3.2 RA test (-.! (-) (-) (-) (-) (-) CRP (mg/dl) 0.03 0.06 0.01 0.07 0.12 ~ 0.02 ANA <40 <40 I <40 <40 <40 <40 ANA pattern (-1 (-) (-) (-) (-) (-) CH50 (CH50/m.l) ;53.0 33.0 42.0 53.0 52.0 45.0 LAK enhancers containing the extract of Lentinus edodes mycelium of t:he present invention showed similar effects to those of IL-2 with no direct toxicity to lymphocytes when i:hey were directly administered to peripheral blood. They are available at far lower cost as compared with IL-2. Therefore, they can be substituted for expensive IL-2 which also has the disadvantage of causing strong side effects.
Formulations containing the extract of Lentinus edodes mycelium of the present invention could remarkably increase :LAK activity with no side effects even by direct administration. 'rhe:refore, formulations containing the extract of Lentinus edodes mycelium of the present invention are useful- as an immunopotentiator capable of increasing LAK activity with a benign side-effect profile by direct administration such as oral administration without using LAK 'therapy.
Claims (11)
1. An LAK activity enhancer containing an extract of Lentinus edodes mycelium.
2. The LAK activity enhancer of claim 1 for enhancing LAK activity by acting on lymphocytes derived from peripheral blood.
3. The LAK activity enhancer of claim 2 containing an extract of Lentinus edodes mycelium at a concentration of 1 µg or more per 10 6 lymphocytes.
4. An LAK activity-enhancing formulation containing the LAK activity enhancer of claim 1.
5. A pharmaceutical or veterinary LAK activity-enhancing formulation comprising the LAK activity enhancer of claim 1 and a pharmaceutically acceptable carrier.
6. The LAK activity-enhancing formulation of claim 4 or 5 for oral administration.
7. The LAK activity-enhancing formulation of claim 4 in the form of a food, drink or feed.
8. The LAK activity-enhancing formulation of claim 4 or 5 for injection or percutaneous administration.
9. The LAK activity-enhancing formulation of any one of claims 4 to 8, wherein said formulation is used for treating tumor and/or cancer.
10. A method for treating tumor and/or cancer comprising administering an effective amount of the LAK
activity-enhancing formulation of any one of claims 4 to 8.
activity-enhancing formulation of any one of claims 4 to 8.
11. A use of the LAK activity enhancer of claim 1 for the preparation of a drug used for treating tumor and/or cancer.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10/353927 | 1998-11-27 | ||
| JP10353927A JP2000159685A (en) | 1998-11-27 | 1998-11-27 | Lak activity potentiator arising from extract from mycelia of lentinus edodes |
| PCT/JP1999/006616 WO2000032212A1 (en) | 1998-11-27 | 1999-11-26 | Lak activity potentiator orginating in shiitake mushroom hyphae extract and lak activity potentiating preparations containing the same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2352512A1 true CA2352512A1 (en) | 2000-06-08 |
Family
ID=18434169
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002352512A Abandoned CA2352512A1 (en) | 1998-11-27 | 1999-11-26 | Lak activity enhancers derived from extract of lentinus endodes mycelium and lak activity-enhancers formulations containing the extract |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JP2000159685A (en) |
| CA (1) | CA2352512A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109481477A (en) * | 2018-12-17 | 2019-03-19 | 上海市农业科学院 | A kind of Preparation method and use of the annesl mycelia mycoderma alcohol extracting thing of mushroom mycelium |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH037236A (en) * | 1989-02-10 | 1991-01-14 | Nippon Chem Res Kk | Agent for inhibiting absorption of herpes virus |
| JP3017630B2 (en) * | 1993-12-17 | 2000-03-13 | 長岡 均 | HIV-type virus activity inhibitor |
-
1998
- 1998-11-27 JP JP10353927A patent/JP2000159685A/en not_active Withdrawn
-
1999
- 1999-11-26 CA CA002352512A patent/CA2352512A1/en not_active Abandoned
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109481477A (en) * | 2018-12-17 | 2019-03-19 | 上海市农业科学院 | A kind of Preparation method and use of the annesl mycelia mycoderma alcohol extracting thing of mushroom mycelium |
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| Publication number | Publication date |
|---|---|
| JP2000159685A (en) | 2000-06-13 |
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