EP1774012A2 - Composes a modulation immunitaire issus de champignons - Google Patents

Composes a modulation immunitaire issus de champignons

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Publication number
EP1774012A2
EP1774012A2 EP05758000A EP05758000A EP1774012A2 EP 1774012 A2 EP1774012 A2 EP 1774012A2 EP 05758000 A EP05758000 A EP 05758000A EP 05758000 A EP05758000 A EP 05758000A EP 1774012 A2 EP1774012 A2 EP 1774012A2
Authority
EP
European Patent Office
Prior art keywords
composition
composition according
polysaccharides
glucose
mannose
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05758000A
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German (de)
English (en)
Inventor
Bjoern Kristiansen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Glycanova As
Original Assignee
MediMush AS
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Filing date
Publication date
Priority claimed from US10/892,393 external-priority patent/US7514085B2/en
Application filed by MediMush AS filed Critical MediMush AS
Publication of EP1774012A2 publication Critical patent/EP1774012A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A61K36/074—Ganoderma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/08—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis for Pneumocystis carinii
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to immune modulating compounds comprising polysaccharides that may be isolated from a liquid culture of fungi.
  • lentinan a polysaccharide based compound described as a beta-(1,3) glucan backbone with beta-(1 ,6) side chains.
  • Solid-state reactors are routinely used for culturing fungi such as Lentinus edodes. This is a technology which is used for many purposes such as composting, production of biological products such as enzymes, soy sauce, acetic acid, and the like.
  • lentinan - Lentinus edodes.
  • the fruiting bodies are harvested, either by hand or mechanically, and are subsequently dried and ground to a powder which can be used as it is, or used in tablets, or sent for further processing such as extraction of lentinan.
  • a polysaccharide product prepared from Lentinus edode is commercially available as "Lentinan for injection” (Eureka Bio-Chemicals Pty, Australia). This product has some immunestimulating effect (see also the examples herein below).
  • compositions with very high immunestimulating activity have not been disclosed. It is therefore an object of the present invention to provide novel compostions, preferably extracellular immune stimulating agents from fungi with high level of immune modulating activity.
  • the present invention discloses compositions comprising polysaccharides with significant immune modulating activity. Interestingly, the present invention discloses that the specific monosaccharide content of a polysaccharide composition is important for the immune modulating activity.
  • a composition comprising one or more polypeptides and a mixture of polysaccharides, wherein the majority of the polysaccharides of the composition, preferably every polysaccharide has a molecular weight of at least 10,000 Da and wherein said mixture of polysaccharides comprises the monosaccharides galactose, mannose, and glucose in the ratio (0- 1.5) : (0-0.5) to (15-35) : (0.5-1.5) to (45-55), preferably in the ratio of about 1 :0 to 25:1 to 50, such as in the ration of 1 :0 to 25:1 to.50.
  • a composition comprising one or more polypeptides and a mixture of polysaccharides, wherein: a) the majority of the polysaccharides of the composition have a molecular weight of at least 30,000 Da and wherein said mixture of polysaccharides comprises the monosaccharides galactose, mannose and glucose in the ratio 1 :0 to 25:1 to 50, or b) the majority of the polysaccharides of the composition have a molecular weight of at least 100,000 Da and wherein said mixture of polysaccharides comprises the monosaccharides galactose, mannose and glucose in the ratio 0 to 0.5:0.5 to 10:0.5 to 50, or c) the majority of the polysaccharides of the composition have a molecular weight of at least 1 ,000 Da and wherein said mixture of polysaccharides comprises the monosaccharides galactose, mannose and glucose in the ratio 1 :0 to 25:1 to 50.
  • compositions according to the invention in the manufacture of a medicament for treatment, optionally prophylactic treatment of an immune compromised condition of an individual in need of such treatment.
  • Polysaccharides covers polysaccharides as well as polysaccharides containing and/or covalently linked to peptides, polypeptides or the like, such as proteopolysaccharides.
  • Polysaccharides comprising monosaccharides A polysaccharide is said to comprise monosaccharides, wherein said monosaccharides are covalently linked to form said polysaccharide. Hydrolysing a polysaccharide will yield the monosaccharides that formed said polysaccharide in free form.
  • the monosaccharide content of a polysaccharide can thus be determined by hydrolysing the polysaccharide and measuring the presence of individual monosaccharides.
  • the monosaccharide content of a mixture of polysaccharides is determined by determining the monosaccharide content of the entire mixture.
  • a polysaccharide or a mixture of polysaccharides are said to comprise galactose, mannose, and glucose in a given ratio, when hydrolysation of said polysaccharide or said mixture of polysaccharide yields galactose, mannose and glucose in said given ratio.
  • Galactose, mannose, and glucose in the ratio 1 :a to b:c to d means that for every part galactose, mannose is present in the range of a to b parts and glucose is present in the range of c to d parts, wherein a, b, c and d indicates numerical values.
  • a polysaccharide mixture comprising galactose, mannose, and glucose in the ratio 1:5 to 25:1 to 50, means that for every part galactose, the polysaccharide mixture comprises in the range of 5 to 25 parts mannose and in the range if 1 to 50 part glucose.
  • Molecular weight Every polysaccharide of a composition is said to have a molecular weight of at least a given value, when said composition has been purified using a filtration step resulting in a molecular weight cut-off of said given value.
  • every polysaccharide of a composition is said to have a molecular weight within a given range, when said composition has been subjected to one or more filtration steps resulting in a lower molecular weight cut-off which is the lower value of the range and an upper molecular weight cut-off which is the upper value of the range.
  • Said filtration step may for example be ultrafiltration, microfiltration, ultracentrifugation or gel filtration.
  • a composition wherein every polysaccharide has a molecular weight of at least a given value or every polysaccharide is said to have a molecular weight within a given range may also be prepared by other methods.
  • Polypeptide covers proteins, peptides and polypeptides, wherein said proteins, peptides or polypeptides may or may not have been post-translationally modified. Post-translational modification may for example be phosphorylation, methylation, glucosylation.
  • compositions according to the present invention comprise one or more polypeptides and a mixture of polysaccharides.
  • Said polypeptides may optionally be covalently linked to polysaccharides. It is however also comprised within the present invention that said polypeptides are either not associated with said polysaccharides or that said polypeptides are associated with said polysaccharides in a non-covalent manner.
  • compositions comprise polysaccharides which may or may not be proteopolysaccharides, or the compositions may comprise a mixture of both.
  • the compositions according to the present invention are isolated or purified compositions, i.e. they have been subjected to one or more purification steps. In a preferred embodiment they have been purified from liquid growth medium of a fungal mycelium using at least one purification step comprising a size fractionation. Methods of purification are described in more detail below.
  • the composition essentially consists of polysaccharides and optionally polypeptides,-more preferably the composition essentially consists of polysaccharides and polypeptides.
  • the composition is a liquid composition, and it is then preferred that the composition essentially consists of polysaccharides and optionally polypeptides dissolved in an aqueous solution optionally comprising salts and buffer.
  • the polysaccharides of the compositions of the invention preferably comprises the monosaccharides glucose, mannose and galactose.
  • polysaccharides and polypeptides that may be comprised within the compositions of the invention include, but are not limited to, ⁇ -(1 ,3), ⁇ -(1 ,6) D-glucans, schizophyllan, grifolan, coriolan and Coriolus versicolor glycosylated polypeptides such as PSK and PSP, polypeptides associated with alpha-mannan such as KS-2, which may be isolated from Lentinus edodes, and reishi, which may be isolated from Ganoderma lucidum; glucuronoxylomannans; mannoglucans; glucomannans; galactoglucan and/or galactoglucomannans.
  • compositions disclosed herein have been produced by a fungus.
  • the compositions have been purified from the extracellular environment of a fungus.
  • the fungus preferably a fungal mycelium, has been cultivated in a liquid growth medium and said composition has been purified from said liquid growth medium.
  • composition of the invention has been produced by a method comprising the steps of i) cultivating a fungus, such as a fungal mycelium, in a liquid growth medium, and ii) isolating the composition from said liquid growth medium
  • fungal mycelium any fungal biomass, which can be grown in a submerged culture.
  • the fungal biomass may be in the form of single hyphae, spores, aggregates of mycelium, and partly differentiated mycelium.
  • the liquid growth medium may be any of the liquid growth media described herein below.
  • the fungus may be any fungus, preferably a fungus forming a fungal mycelium, more preferably the fungus is a filamentous fungus. Even more preferably, the fungus may be selected from the group consisting of Agaricus bisporus, Cordiceps sinensis, Flammulina velutipes, Ganoderma lucidum, Grifola frondosa, Lentinus edodes, Pleurotus ostreatus, Schizophyllum commune, Sclerotina sclerotium, Trametes (Coriolus) versicolor, Tremella fuciformis, Agaricus blazei, Agrocybe aegerita, Agrocybe cylindracea, Albatrellus confluens, Armilla ⁇ ella mellea, Au ⁇ cula ⁇ a au ⁇ cula-judae, Auricularia polytrichia, Collybia maracula, Cordiceps militari, Dendrop
  • Ganoderma applanatum Ganoderma tsugae, Hericium erinaceus, Hypsizygus marmoreus, lnonotus obliquus, Laetiporus sulphurous, Lenzites betulinus, Leucopaxilllus giganteus, Lyophyllum cinerascens, Omphalina epichysium, Oudemansiella mucida, Panellus serotinus, Piptoporus betulinus, Phellinus linteus, Phellinus pini, Pholiota nameko, Pleurotus citrinopileatus, Pleurotus pulmonarius, Sarcedon asparatus, Trametes suavolens, Volvariella volvacea and Wolfiporia cocos.
  • the fungus is selected from the group consisting of Agaricus bisporus, Cordiceps sinensis, Flammulina velutipes, Ganoderma lucidum, Grifola frondosa, Lentinus edodes, Pleurotus ostreatus, Schizophyllum commune, Sclerotina sclerotium, Trametes (Coriolus) versicolor and Tremella fuciformis
  • the fungus is Agaricus blazei.
  • said fungus is Ganoderma, such as Ganoderma lucidum.
  • the fungus is an Agaricus fungus, such as selected from the group consisting of: A. blazei, A. blazei Murill, A. bisporus, A. hortensis, A. campestris.
  • the fungus belongs to the class of basidiomycetes, such as a fungus of the genus Lentinus, such as Lentinus edodes.
  • Lentinus pygmaeus Colenso Lentinus zelandicus Sacc. & Cub. (1887); Lentinus sajor-caju (Fr.) Fr.; Lentinus squarrulosus Mont; Lentinus strigosus (Schwein.) Fr. (1825); Lentinus suffrutescens (Brot.) Fr. (1825); and Lentinus tuber-regium Fr.; Lentinus zelandicus Sacc. & Cub. (1887) (Ref: http://nzfungi . landcareresearch .co. nz) . Modulation of the immune system
  • compositions according to the invention are preferably immune modulating, preferably, the compositions are immune stimulating.
  • the stimulation of the immune system can be demonstrated by e.g. increased antibody production, by activation of helper T-cells, or by increased production of interleukins such as lnterleukin 1 and lnterleukin 2.
  • any assay known to the skilled person, which is suitable for testing whether a composition is immune modulating may be employed to test whether a composition of the present invention is immune modulating.
  • Such an assay may be an in vitro or an in vivo assay.
  • the composition according to the present invention is capable of inducing IL-1 production from at least one kind of IL-1 producing cells in an in-vitro assay.
  • the cells may be any IL-1 producing cells, such as P388 mouse macrophage cells.
  • said cell is a IL-1 beta producing cell.
  • IL-1 production may be determined using any suitable assay.
  • assays involving the use of specific IL-1 antibodies, such as specific IL1- ⁇ and/or IL1- ⁇ antibodies are useful.
  • Such assays may for example be Western blotting, ELISA or similar assays.
  • the assay may be performed as described in example 4.
  • the assay is preferably performed using a specific composition as reference.
  • the composition is capable of inducing production of at least 1.5, preferably at least 2, such as at least 4, for example at least 6, such as at least 8, for example at least 10, such as at least 15, for example at least 20, such as at least 30, for example at least 40 times more IL1- ⁇ , than the amount of IL1- ⁇ induced using the commercially available Lentinan for injection (Eureka Bio-Chemicals Pty, Little Collins Street. Melbourne 3000, Australia) in a reference experiment performed in parallel..
  • the composition is capable of inducing production of at least 1.5, preferably at least 2, such as at least 4, for example at least 6, such as at least 8, for example at least 10, such as at least 15, for example at least 20 times more IL1- ⁇ , than the amount of IL1- ⁇ induced using Lentinan for injection from Eureka Biochemicals Pty. in a reference experiment performed in parallel.
  • the aforementioned assays are performed as described in example 4. It is most preferred that the composition induced production of both IL1 ⁇ and IL1 ⁇ as described above.
  • the composition is capable of enhancing antibody production in a mammal, when administered to said mammal.
  • the mammal may for example be a mouse, rat, rabbit or even a human being.
  • an assay is performed by administering the composition to a mammal prior to and simultaneously with administration of an antigen, optionally in the presence of an adjuvant.
  • the composition is administered in the range of 1 to 30 days, preferably in the range of 1 to 10 days, more preferably in the range of 1 to 3 days prior to administration of the antigen. Subsequently, antibody production in the mammal may be determined.
  • the composition is preferably capable of inducing production of at least 1.5, more preferably at least 2, even more preferably at least 2.5, such as at least 3, for example at least 4, such as 6 times more antibody compared to the amount of antibody produced without administration of the composition.
  • An example of such an assay is outlined in example 6.
  • composition is immune modulating in more than one assay system, such as in a combination of any of the assay systems described herein above.
  • compositions of the present invention act to increase TNF-alpha production, such as may be measured by the assay described in Example 9.
  • composition according to the present invention preferably comprises polysaccharides, wherein the majority of the polysaccharides, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, yet more preferably at least 90%, even more preferably at least 95%, yet more preferably essentially all polysaccharides, most preferably every polysaccharide has a molecular weight above 10,000 Da, preferably above 30,000 Da, more preferably above 40,000 Da, even more preferably above 50,000 Da, for example at least 100,000 Da, such as at least 300,000 Da, for example at least 1,000,000 Da.
  • the majority of polysaccharides preferably every polysaccharide of the composition has a molecular weight within the range of 10,000 to 3,000,000 Da., for example within the range of 30,000 to 3,000,000, such as within the range of 40,000 to 3,000,000, for example within the range of 50,000 to 3,000,000, such as in the range of 50,000 to 100,000, for example in the range of 100,000 to 300,000, such as in the range of 300,000, to 1,000,000, for example in the range of 1 ,000,000 to 3,000,000.
  • the composition has been purified by a method involving at least one size fractionation step.
  • the composition has been purified using at least one size fractionation step wherein molecules, such as polysaccharides with a nominal molecular weight above a given cut-off are separated from molecules, such as polysaccharides with a nominal molecular weight below said cut-off.
  • molecules such as polysaccharides with a nominal molecular weight above a given cut-off are separated from molecules, such as polysaccharides with a nominal molecular weight below said cut-off.
  • the size fractionation is ultrafiltration or microfiltration a membrane with said cut-off may be used.
  • the size fractionation is gel filtration a gel with said molecular weight cut off may be chosen or a particular elution fraction may be used.
  • the larger molecular weight fraction is used, wherein the cut-off preferably is 10,000 Da, more preferably 30,000 Da, even more preferably 40,000 Da, yet more preferably 50,000 Da.
  • the composition has been purified by a method involving one or more size fractionation steps, wherein a resulting fraction comprises polysaccharides with a nominal molecular weight below a given cut-off and above a given cut-off.
  • a resulting fraction comprises polysaccharides with a nominal molecular weight below a given cut-off and above a given cut-off.
  • the size fractionation is ultrafiltration or microfiltration
  • first one fractionation step using a membrane with the lower molecular weight cut off may be performed.
  • the larger molecular weight fraction may be collected and subjected to a second ultrafiltration or microfiltration using a membrane with the upper molecular weight cut off.
  • the lower molecular weight fraction may be collected.
  • gel filtration a particular elution fraction may be used.
  • ultracentrifugation is used, a membrane with the lower molecular cut-off and a membrane with the upper molecular weight cut-off may be used.
  • composition according to the present invention comprises a mixture of polysaccharides, wherein said mixture comprises the monosaccharides galactose, mannose, and glucose in the ratio 1:5 to 25:1 to 50.
  • the ratio reflects the ratio within the entire mixture of polysaccharides. It is thus feasible that each individual polysaccharide within the mixture comprises a different ratio of the monosaccharides.
  • the ratio may in general be determined by degrading the entire mixture of polysaccharides into monosaccharides and subsequently determining the concentration of each of said monosaccharides.
  • Polysaccharides may be degraded to their constituent monosaccharides by hydrolysis, for example by hydrolysis in a strong acid, such as HCI.
  • the hydrolysate may be analysed by any conventional method available to the skilled person, for example by HPLC, mass spectrometry or NMR.
  • polysaccharides comprise the mono ⁇ saccharides glucose and mannose. In another embodiment of the invention the polysaccharides comprise the monosaccharides glucose and galactose. In a preferred embodiment of the invention, the polysaccharides of the composition comprise the monosaccharides galactose, mannose and glucose.
  • the mixture of polysaccharides comprises in the range of 5 to 25, preferably in the range of 5 to 20, more preferably in the range of 5 to 17, even more preferably in the range of 6 to 15, yet more preferably in the range of 7 to 14, such as in the range of 10 to 17, for example in the range of 11 to 16, such as in the range of 12 to 15, for example in the range of 13 to 14, such as approximately 13.4+/-0.4, for example 13.4+/-0.4 parts mannose for every part galactose.
  • the mixture of polysaccharides comprises in the range of 1 to 50, preferably in the range of 1 to 40, more preferably in the range of 1 to 30, even more preferably in the range of 1 to 25, yet more preferably in the range of 1 to 20, even more preferably in the range of 2 to 15, yet more preferably in the range of 2 to 14, such as in the range of 8 to 17, for example in the range of 9 to 16, such as in the range of 10 to 15, for example in the range of 11 to 14, such as approximately 12.6+/-1.3, for example 12.6+/-1.3 parts glucose for every part galactose.
  • the mixture comprises a ratio of mannose to galactose as indicated herein above and a ratio of glucose to galactose in a ratio as indicated herein above.
  • the polysaccharides comprise mannose and glucose it is preferred that they comprise in the range of 0.1 to 30, such as in the range of 0.1 to 0.25, for example in the range of 0.25 to 0.5, such as in the range of 0.5 to .75, for example in the range of 0.75 to 1 , such as in the range of 1 to 5, for example in the range of 5 to 10, such in the range of 10 to 20, for example in the range of 20 to 30 parts glucose for every part mannose.
  • the polysaccharides comprise in the range of 0.5 to 2 parts glucose for every part mannose, more preferably 13.4 +/-0.4 parts mannose 12.6 +/--1.3 part glucose.
  • the composition comprises a mixture of polysaccharides comprises the monosacharides galactose, mannose, and glucose in the ratio 1:5 to 25:1 to 50, more preferably 1 :13.4 +/- 0.4:12.6 +/- 1.3.
  • the composition comprises a mixture of polysaccharides, wherein the polysaccharides within said mixture having a molecular weight in the range of 50,000 to 100,000 comprises in the range of 3 to 15, preferably in the range of 4 to 14, more preferably in the range of 5 to 13, even more preferably in the range of 6 to 12, yet more preferably in the range of 7 to 11 , even more preferably in the range of 7.9 to 9.9, for example approximately 8.9 parts mannose for every part galactose.
  • the polysaccharides having a molecular weight in the range of 50,000 to 100,000 comprises in the range of 1 to 5, preferably in the range of 2 to 4, even more preferably in the range of 2.5 to 3.5, such as approximately 2.9 parts glucose for every part galactose. It is preferred that the polysaccharides of said composition having a molecular weight in the range of 50,000 to 100,000 Da comprise the monosacharides galactose, mannose, and glucose in the ratio 1:4 to 14:1 to 5, preferably the ratio is approximately 1 :8.9:2.9.
  • the composition comprises a mixture of polysaccharides, wherein the polysaccharides within said mixture having a molecular weight in the range of 100,000 to 300,000 comprises in the range of 3 to 15, preferably in the range of 3 to 14, more preferably in the range of 3 to 13, even more preferably in the range of 3 to 12, yet more preferably in the range of 4 to 11 , even more preferably in the range of 5 to 10, yet more preferably in the range of 6.3 to 8.3, for example approximately 7.3 parts mannose for every part galactose.
  • the polysaccharides having a molecular weight in the range of 100,000 to 300,000 comprises in the range of 1 to 5, preferably in the range of 2 to 4, even more preferably in the range of 2.5 to 3.5, such as approximately 2.9 parts glucose for every part galactose. It is preferred that the polysaccharides of said composition having a molecular weight in the range of 50,000 to 100,000 Da comprise the monosacharides galactose, mannose, and glucose in the ratio 1 :3 to 13:1 to 5, preferably the ratio is approximately 1 :7.3:2.9.
  • the composition comprises a mixture of polysaccharides, wherein the polysaccharides within said mixture having a molecular weight in the range of 300,000 to 1 ,000,000 comprises in the range of 3 to 16, preferably in the range of 5 to 15, more preferably in the range of 5 to 14, even more preferably in the range of 6 to 13, yet more preferably in the range of 7 to 12, even more preferably in the range of 8.9 to 10.9, for example approximately 9.9 parts mannose for every part galactose.
  • the polysaccharides having a molecular weight in the range of 300,000 to 1 ,000,000 comprises in the range of 1 to 5, preferably in the range of 2 to 4, even more preferably in the range of 2.5 to 3.5, such as approximately 3.1 parts glucose for every part galactose. It is preferred that the polysaccharides of said composition having a molecular weight in the range of 300,000 to 1 ,000,000 Da comprise the monosacharides galactose, mannose, and glucose in the ratio 1 : 5 to 15:1 to 5, preferably the ratio is approximately 1:9.9:3.1.
  • the composition comprises a mixture of polysaccharides, wherein the polysaccharides within said mixture having a molecular weight of at least 1 ,000,000 comprises in the range of 3 to 17, preferably in the range of 4 to 16, more preferably in the range of 5 to 15, even more preferably in the range of 6 to 14, yet more preferably in the range of 7 to 13, even more preferably in the range of 8 to 12, even more preferably in the range of 9.3 to 11.3, for example approximately 10.3 parts mannose for every part galactose.
  • the polysaccharides having a molecular weight of at least 1 ,000,000 comprises in the range of 1 to 5, preferably in the range of 2 to 4, even more preferably in the range of 2.5 to 3.5, such as approximately 2.9 parts glucose for every part galactose. It is preferred that the polysaccharides of said composition having a molecular weight in the range of at least 1 ,000,000 Da comprise the monosacharides galactose, mannose, and glucose in the ratio 1 :4 to 1 :5 to 15:1 to 5, preferably the ratio is approximately 1:10.3:2.9.
  • the polysaccharides according to the present invention have a molar ratio of galactose:mannose:glucose of 1 : 10 to 20 : 30 to 50, such as 1 : 12 to 18 : 35 to 45; for example 1 : 14 to 16 : 38 to 42, such as 1 : about 15 : about 40, for example 1 : 15 : 40.
  • a composition the polypeptides comprise the monosaccharides galactose, mannose and glucose in the ratio (galactose : mannose : glucose) of 1 : 0 to 25 : 1 to 50, such as 1 : 10 to 20 : 30 to 50, such as 1 : 12 to 18 : 35 to 45; for example 1 : 14 to 16 : 38 to 42, such as 1 : about 15 : about 40, for example 1 : 15 : 40.
  • the polysaccharides according to the present invention have a molar ratio of galactose:mannose:glucose of 1 : 0.5 to 5 : 6 to 12, such as 1 : 1 to 4 : 7 to 11 ; for example 1 : 1.5 to 3.5 : 7.5 to 10, such as 1 : 2.0 to 3.0 : 7.5 to 9.5, for example 1 : 2.2 to 2.8 : 8.0 to 9.0, such as 1 : about 2.5 : 8.0 to 9.0, for example 1 : 2.5 : 8.0 to 9.0, such as 1 : 2.5 : 8.6.
  • composition of the present invention have a molar ratio of galactose:mannose:glucose of 1:12:40 and 1 :3:5.
  • the sugar composition is obtainable from Agaricus blazei and comprises 10-20 parts mannose, 30-50 parts glucose and 0-2 parts galactose, such as e.g. 11-17 parts mannose, 35-45 parts glucose and 0-1 parts galactose. More preferably, the sugar composition is obtainable from Agaricus blazei and comprises about 15 parts mannose, about 40 parts glucose and about 1 part galactose, wherein "about” signifies within 20 % difference from the stated value, such as within 10 % difference from the stated value., such as within 5 % difference from the stated value.
  • the sugar composition is obtainable from Ganoderma and comprises 0.5-5 parts mannose, 4-15 parts glucose and 0-2 parts galactose, such as e.g. 2-3 parts mannose, 7-9.5 parts glucose and 0.5-1.5 parts galactose. More preferably, the the sugar composition is obtainable from Ganoderma and comprises about 2.5 parts mannose, about 8.6 parts glucose and about 1 part galactose, wherein "about” signifies within 20 % difference from the stated value, such as within 10 % difference from the stated value, such as within 5 % difference from the stated value.
  • Determination of the monosaccharide content of polysaccharides with a molecular weight within a given range may be determined by fractionating the composition according to size for example by ultrafiltration, microfiltration, ultracentrifugation or gelfiltration. For example this may be done as described in example 2.
  • the mixture of polysaccharides comprises the monosaccharides galactose, mannose, and glucose in the ratio (0-1.5) : (0-0.5) to (15-35) : (0.5-1.5) to
  • (45-55) such as e.g . any of the following: (0-1.5) : (0-0.5) to (15-35) : (0.5-1.5) to (45-55),
  • the sugar composition of the composition of the present invention comprises 1-25 parts mannose, 5-45 parts glucose and 0-1.5 parts galactose; (by “part” is meant herein the relative molar ratio, i.e. for every 1-25 moles mannose, there are 5-45 moles of glucose and 0-1.5 moles of galactose), thus the sugar composition may comprise A), B) and C), as follows:
  • a composition comprising one or more polypeptides and a mixture of polysaccharides, wherein: a) the majority of the polysaccharides of the composition have a molecular weight of at least 30,000 Da and wherein said mixture of polysaccharides comprises the monosaccharides galactose, mannose and glucose in the ratio 1 :0 to 25:1 to 50, or b) the majority of the polysaccharides of the composition have a molecular weight of at least 100,000 Da and wherein said mixture of polysaccharides comprises the monosaccharides galactose, mannose and glucose in the ratio 0 to 0.5:0.5 to 10:0.5 to 50, or c) the majority of the polysaccharides of the composition have a molecular weight of at least 1 ,000 Da and wherein said mixture of polysaccharides comprises the monosaccharides galactose, mannose and glucose in the ratio 1 :0 to 25:1 to 50.
  • the majority of the polysaccharides of the composition have a molecular weight of at least 10,000 Da, preferably at least 30,000 Da, and said mixture of polysaccharides comprises the monosaccharides galactose, mannose and glucose in the ratio 1 :0 to 25:1 to 50, such as any of the following ratios:
  • the majority of the polysaccharides of the composition have a molecular weight of at least 1,000 Da, such as at least
  • said mixture of polysaccharides comprises the monosaccharides galactose, mannose and glucose in the ratio 0 to 0.5:0.5 to 10:0.5 to 50, such as any of the following ratios:
  • said ratio is about 0: 1 : 18 or about 0: 1 : 1.
  • the majority of the polysaccharides of the composition have a molecular weight of at least 500 Da, such as at least 800 Da, such as at least 1 ,000 Da, for example at least 5,000 Da, for example at least 10,000 Da, such as at least 20,000 Da, for example at least 40,000 Da, such as at least 50,000 Da, for example at least 60,000 Da 75,000 Da, for example at least
  • said mixture of polysaccharides comprises the monosaccharides galactose, mannose and glucose in the ratio 1 :0 to 25:1 to 50, such as any of the following ratios:
  • said ratio is about 1:12:40 or about 1:3:5.
  • composition according to the invention preferably comprises polypeptides.
  • polypeptide as used herein covers both proteins, peptides and polypeptides. Said polypeptides may be in free form, they may be covalently linked to a polysaccharide or they may be non-covalently associated with a polysaccharide or a mixture of the aforementioned.
  • the composition comprises sufficient polypeptide in order to allow for oral administration of the composition. If the composition comprises too little polypeptide, then no or little immune modulation is obtained in an individual after oral administration of the composition to said individual. It is therefore preferred that the composition of the invention comprises at least 10 ⁇ g/L, more preferably at least 20 ⁇ /L, even more preferably at least 25 ⁇ g/L, for example in the range of 10 to 1000 ⁇ g/L, such as in the range of 20 to 1000 ⁇ g/L, for example in the range of 25 to 1000 ⁇ g/L, such as in the range of 25 to 100 ⁇ g/L, for example in the range of 25 to 35 ⁇ g/L polypeptide, preferably soluble polypeptide.
  • composition comprising the aforementioned concentration of polypeptide, comprises in the range of 0.1 to 2, more preferably in the range of 0.5 to 1.5, even more preferably around 1 mg/ml polysaccharide. If the composition comprises more or less polysaccharide it is preferred that the amount of polypeptide is proportionally reduced or enhanced.
  • compositions of the invention are prepared by a method comprising the steps of
  • Cultivating the fungus in a liquid growth medium in general involves dissolving nutrient compounds required for growth of said fungus in water, transferring the solution to a bioreactor and inoculating the bioreactor with cells or spores of the fungus, such as a fungal mycelium, or fractions thereof, to be cultivated. This is done under sterile conditions and with control of the environment in order to give the fungus a suitable chemical and physical environment. Cultivating fungi in liquid growth medium is also termed "liquid state" cultivation.
  • the medium with the fungal biomass is preferably agitated to reduce the occurrence of gradients and to ensure oxygen availability to the submerged cells.
  • oxygen may be supplied to the liquid medium and the level of dissolved oxygen may be controlled by known methods.
  • the liquid growth medium is an aqueous solution, preferably sterile water, comprising nutrient compounds.
  • the liquid medium supports fungal growth and preferably stimulates the production of extracellular compounds, such as immune modulating agents.
  • the liquid growth medium may comprise one or more typical ingredients required for growth of microbial organisms such as malt extract, yeast extract, peptone, glucose, sucrose, sucrose, salts providing phosphate, magnesium and potassium, corn-steep liquor and vitamins such as thiamine. More preferably, the medium comprises sucrose, corns steep liquor, phosphate and magnesium for mycelium growth and production of polysaccharides.
  • fungal mycelium such as Lentinus edodes mycelium from agar plates containing for example malt extract, yeast extract, peptone and glucose can be used.
  • Fungi can initially be cultivated on agar plates comprising the above nutrient compounds supporting the growth of the fungus. The plates are inoculated with mycelium and incubated at least until a visible growth is evident on the plates.
  • this usually can take from about 7 days to about 24 days or from about 10 to 30 days, typically 14 days or up to 20 days, at a temperature in the range of from 18 to 32 0 C, preferably in the area of from 22 to 3O 0 C, such as a temperature of about 23 0 C to 27 0 C, such as around 25°C.
  • inoculation of the growth medium can be carried out by using mycelium from a fermentation broth in e.g. a shake flask medium comprising nutrient compounds supporting cell growth.
  • Shake flasks for cultivating fungal mycelium can initially be inoculated with the mycelium which is cultivated on agar plates. The mycelium is taken from the plates and transferred aseptically to shake flasks containing sterile water comprising dissolved nutrient compounds and nutrient salts supporting the growth of the fungal mycelium.
  • a typical growth medium contains sucrose, corn steep liquor, phosphate and a magnesium. The amount of inoculation material which gives the highest production of extracellular lentinan can be selected following initial experiments.
  • the shake flasks can be incubated by shaking for 6 to 21 days, preferably from 7 to 18 days, more preferably from 8 to 14 days at a temperature in the range of from 18 to 32 0 C, preferably in the area of from 22 to 3O 0 C, such as a temperature of about 23 0 C, for example 24 0 C, such as 25 0 C 1 for example 26 0 C, such as 27 0 C, for example 28 0 C, such as 29 0 C, for example 3O 0 C.
  • the shake flasks may also be incubated from 8-25 days, more preferably from 10-20 days, more preferably from 12-18 days.
  • the temperature may also be from 18 to 37°C, preferably from 23 to 32 0 C such as about 25°C.
  • the content of the shake flasks can be used for inoculating a bioreactor.
  • the reactor comprises a sterile solution of nutrient compounds and nutrient salts in water for mono-culture cultivation of basidiomycete fungal mycelium, or fractions thereof, such as Lentinus fungal mycelium, such as Lentinus edodes.
  • the bioreactor fermentation period is typically in the range of from 50 hours to 300 hours, preferably in the range of from 80 hours to 270 hours, and the temperature is kept constant in the range of 18 to 32 0 C, preferably in the area of from 22 to 31 0 C, such as a temperature of about 23 0 C, for example 24 0 C, such as 25 0 C, for example
  • the temperature may also be from 18 to 37 0 C, preferably from 23 to 32°C such as about 25°C.
  • the reactor is fitted with an inlet for supplying air to the fermentation broth, and the fermentation broth is preferably kept under continuous agitation either as a result of the addition of air, or by means of a mixer device suitable for providing a good mixing of the content of the reactor.
  • the pH of the growth medium is adjusted to from about 3 to about 7, such as a pH of from about 4.5 to about 6.5, for example a pH of about 6, before the growth medium is inoculated with fungal mycelium, or fractions thereof, such as L. edodes mycelium.
  • pH may be dropped naturally during the course of the fermentation, or controlled at a particular value in the range pH 3 to 7, using addition of suitable pH-control agents, such as acid and base.
  • the temperature of the growth medium is preferably in the range of from 18 to 32 0 C, preferably in the area of from 22 to 31 0 C, such as a temperature of about 23 0 C, for example 24 0 C, such as 25 0 C, for example 26 0 C, such as 27 0 C, for example 28 0 C, such as 29 0 C, for example 3O 0 C.
  • the temperature may also be from 18 to 37 0 C, preferably from 23 to 32°C such as about 25 0 C.
  • Samples can be obtained from the bioreactor and analysed for biomass, metabolic products and nutrient compounds, the determinations of which can assist the operator of the bioreactor in the running of the fermentation process. Typical analyses routinely carried out are determination of biomass, residual sugar concentration and extracellular polysaccharide concentration. A person skilled in the art knows the methods for analysis which can be employed in this respect.
  • the method for preparing the compositions according to the invention involves a step of purifying the extracellular fraction of the liquid growth medium from the fungal mycelium.
  • the extracellular fraction of the liquid fermentation medium is also termed the supernatant and this fraction can be separated from the fungal mycelium by e.g.
  • the term "essentially without any fungal mycelium present therein” shall denote that the concentration of fungal mycelium, including fractions thereof, has been reduced at least by a factor of 10 3 , such as reduced by a factor of at least 10 4 , for example a factor of at least 10 5 , such as reduced by a factor of at least 10 6 .
  • the purification comprises at least one size fractionation step.
  • this size fractionation step is performed on the extracellular fraction.
  • This size fractionation step may ensure that every polysaccharide of the composition has a molecular weight of at least a given value (see also herein above).
  • the size fractionation step may be any size fraction known to the skilled person, for example ultracentrifugation, ultrafiltration, microfiltration or gelfiltration.
  • the composition is purified from a liquid growth medium by a method involving one or more purification steps selected from the group consisting of ultracentrifugation, ultrafiltration, microfiltration and gelfiltration.
  • the purification step(s) are selected from the group consisting of ultrafiltration, microfiltration and ultracentrifucation, even more preferably from the group consisting of ultrafiltration and microfiltration.
  • Ultrafiltration is a membrane process where the membrane fractionates components of a liquid according to size.
  • the membrane configuration is normally cross-flow wherein the liquid containing the relevant components are flowing across the membrane. Some of the liquid, containing components smaller than the nominal pore size of the membrane will permeate through the membrane. Molecules larger than the nominal pore size will be retained.
  • the desired product may be in the retentate or the filtrate. If the ultrafiltration is performed in order to prepare a composition, wherein every polysaccharide within said composition has a molecular weight above a given value, the desired product is in the retentate. If a serial fractionation is made, the product may be in the retentate or filtrate.
  • Microfiltration is a membrane separation process similar to UF but with even larger membrane pore size allowing larger particles to pass through.
  • Gel filtration is a chromatographic technique in which particles are separated according to size.
  • the filtration medium will typically be small gel beads which will take up the molecules that can pass through the bead pores. Larger molecules will pass through the column without being taken up by the beads.
  • Gel-filtration, ultrafiltration or microfiltration may for example be performed as desribed in R Hatti-Kaul and B Mattiasson (2001), Downstream Processing in Biotechnology, in Basic Biotechnology, eds C Ratledge and B Kristiansen, Cambridge University Press) pp 189.
  • compositions according to the invention are described in e.g. examples 1 , 7 and 8.
  • the method therefore does not involve an alcohol precipitation step, more preferably the method does not involve a precipitation step.
  • compositions of the invention in a method for stimulating the immune system of an individual in need of such stimulation.
  • the composition may be administered e.g. orally or subcutaneously to the individual in a pharmaceutically effective amount capable of stimulating the immune system of the individual.
  • the stimulation of the immune system can be demonstrated by e.g. increased antibody production, by activation of helper T-cells, or by increased production of interleukins such as lnterleukin 1 and lnterleukin 2.
  • compositions of the invention in the manufacture of a medicament for treating an immune compromised condition in an individual in need of such treatment.
  • An immune compromised condition in an individual may be demonstrated e.g.
  • the immune compromised condition may be any of those disclosed below.
  • the treatment may be prophylactic, ameliorating or curative.
  • Insufficient amount and “decreased production” as used herein above shall denote such amounts and productions which a medical expert considers as being below a predetermined level or value normally associated with a healthy individual.
  • the amount and/or production will generally depend on factors such as age, general physical condition, and the like. For this reason a predetermined level or value shall be determined on an individual basis by a medical expert.
  • One indication of an immune compromised condition in an individual is a gradually decreasing number of antibodies, a gradually decreasing number of CD4 (positive) cells, or a gradually decreasing number of T-helper cells per unit (blood) sample volume measured over time, such as days, weeks, months or years.
  • the present invention relates to pharmaceutical compositions comprising the composition according to the invention and pharmaceutically acceptable carrier(s).
  • the pharmaceutical compositions may be prepared by conventional techniques, e.g. as described in Remington: The Science and Practice of Pharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton, Pa.
  • the compositions may appear in conventional forms, for example capsules, tablets, lozenges, powders, syrups, solutions or suspensions.
  • the invention relates to a method of treatment of an individual diagnosed with an immune compromised condition, said method comprising the steps of administering to said individual the composition according to the invention or the pharmaceutical composition according to the invention in an amount effective in treating said immune compromised condition.
  • the administered amount may in be an amount effective in prophylactically treating said immune compromised condition.
  • a method of treatment of an individual diagnosed with or at risk of contracting acquired immunodeficiency syndrome comprising the steps of administering to said individual the composition according to the invention or the pharmaceutical composition according to the invention in an amount effective in treating or prophylactically treating said syndrome.
  • the immune compromised condition may be selected from the group consisting of an infectious disease, a parasitic disease, haemophilus meningitis, pneumococcal meningitis, streptococcal meningitis, staphylococcal meningitis, meningitis due to other organisms, encephalitis, viral pneumonia, pneumococcal pneumonia, other bacterial pneumonia, pneumonia due to other specified organisms except bacteria, bronchopneumonia, organism unspecific pneumonia, influenza, unspecified diarrhea, hepatitis unspecified, acute and subacute necrosis of the liver, chronic hepatitis, and abscess of liver.
  • the immune compromised condition may be an infectious or parasitic disease caused by, or selected from, cholera, salmonella, shigellosis, Escherichia coli, intestinal infection due to other specified bacteria, Clostridium difficile, viral gastroenteritis, infectious colitis, enteritis and gastroenteritis, infectious diarrhea, tuberculosis, listeriosis, pasteurellosis, mycobacterium, diphtheria, pertussis, meningococcus, streptococcus septicaemia, staphylococcus septicaemia, pneumococcal septicaemia, septicaemia due to anaerobes, septicaemia due to other gram-negative organisms, actinomycotic infection, gas gangrene, toxic shock syndrome, necrotizing faciitis, Friedlander's bacillus, haemophilus influenzae, pseudomonas, AIDS/HIV infections, acute poliomyelitis, Creutzfeldt-Jacob disease, suba
  • the individual may be a mammal, such as a human being.
  • compositions of the invention may be administered using any suitable administration form; usually however, administration will be oral or parenteral. Oral administration in the form of a syrup comprising the composition and/or a capsule containing a syrup comprising the composition or in a powder form of the composition is preferred.
  • the dosage requirements will vary with the particular composition employed, the route of administration and the particular individual being treated. Ideally, an individual to be treated by the present method will receive a pharmaceutically effective amount of the compound in the maximum tolerated dose.
  • the daily oral dosage regimen may be about 0.001 to about 100 mg/kg, preferably in the range of 0.01 to 50 mg/kg, more preferably in the range of 0.1 to 10, even more preferably in the range of 1 to 2 mg/kg of total body weight.
  • the optimal quantity and spacing of individual dosages of the composition will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optimums can be determined by conventional techniques.
  • the optimal course of treatment i.e., the number of doses of the composition given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.
  • compositions and the pharmaceutical compositions according to the invention may also form part of a kit comprising said compositions and a dosage regime instruction with guidelines for dose and time administration.
  • the composition of the present invention is used in the manufacture of a functional food, thus in one embodiment of the present invention, any of the compositions described herein may be comprised in a functional food or nutrition supplement, preferably suitable for human beings.
  • Said functional food is preferably a survival enhancing, longevity enhancing, health enhancing and/or a modulator of a microbial population. More preferably, said functional food is health enhancing and/or a modulator of a microbial population. It is preferred that the functional food is suitable for at least weekly oral intake, such as for daily oral intake.
  • said functional food product may be suitable for use in parenteral or enteral nutrition, preferably in combination with formulations comprising other nutrients known to one skilled in the art.
  • Products according to the invention may be used for promoting health of human beings, for example for maintaining, strengthening or promoting bone or cardiovascular health.
  • the functional food can be used for the prevention or reduction of osteoporosis.
  • regular consumption of said functional food such as for example once a day, twice a day, or three times a day, leads to a reduction of the risk of diseases such as colds, coughs and reduces tiredness and fatigue.
  • the functional food is preferably ingested by a human as an ingredient of his or her daily diet.
  • a liquid vehicle such as water, milk, vegetable oil, juice and the like, or with an ingestible solid or semi-solid foodstuff.
  • the present invention thus relates to a method of producing a functional food composition, comprising mixing any of the compositions described herein (for example obtained from Lentinus fungus) with a foodstuff.
  • said functional food product may be selected from the group of meal replacers, dietary supplements, ice-cream, sauces, dressing, spreads, bars, sweets, snacks, cereals and beverages.
  • said functional food is dietary supplement, preferably suitable for ingestion in pill, capsule, tablet or liquid form.
  • products according to the invention are prepared whereby any of the compocsitions described herein (such as from Lentinus) is added to the food product such that the level of the composition is between 5 to 5000 mg per 100 g product.
  • the functional food is a dairy product.
  • said functional food may for example be selected from any of the following: cultured dairy products, yogurts, cottage cheese, cream cheese, dairy dips, sour cream, milkshakes, Butter, Margarine, Low-fat spreads, Cheese, Cottage cheese, Cheese spread, Cheese "strings” for children.
  • said dairy product is a cheese-based product, such as selected from low-fat cheese, hard cheese, soft cheese, cottage cheese, cheese spread, cheese "strings” for children or cheese slices suitable for sandwiches.
  • said dairy product is a yoghurt-based product, such as selected from a set yoghurt, a runny or pourable yoghurt, a yoghurt-based carbonated drink, a drinking or drinkable yoghurt, a low-fat yoghurt.
  • Said yoghurt-based product may for example be fermented with Lactobacillus bulgaricus and/or Streptococcus thermophilus.
  • said dairy product is a cultured dairy product, such as a cultured fluid (for example drinkable yogurt/yogurt smoothies, kefir, probiotic shots); a non-drinkable yogurt (for example in a cup or tubes); and/or another non-pourable cultured dairy product (for example cottage cheese, cream cheese, dairy dips or sour cream).
  • a cultured fluid for example drinkable yogurt/yogurt smoothies, kefir, probiotic shots
  • a non-drinkable yogurt for example in a cup or tubes
  • another non-pourable cultured dairy product for example cottage cheese, cream cheese, dairy dips or sour cream.
  • said dairy product is another type of dairy product, such as selected from the group consisting of: refrigerated dips and sour cream, ice cream, cream, low-fat cream-replacement, fermented milk such as kefir, fermented beverages, such as drinkable yoghurt and kefir.
  • the functional food according to the present invention is a health drink.
  • Said health drink is in one embodiment fruit juice-based, which may be concentrated as a "squash", to be diluted to taste.
  • Said fruit juice or squash preferably comprises concentrated fruit juice.
  • Preferred fruit juices include, but are not restricted to, citrus fruit juices such as orange, grapefruit, lemon or lime, or combinations thereof.
  • said fruit juice or squash comprises (preferably concentrated) berry juice(s), such as from raspberries, strawberries, blackberries, loganberries, cranberries, redcurrants, blackcurrants, blueberries, or combinations thereof, and/or combinations with citrus fruit juices.
  • said fruit juice or squash comprises juice(s) from one or more of Pineapple, Passion Fruit, Mango, apple, pear, apricot, Pomegranate, guava, tomato and/or combinations with any other types of fruit juices.
  • Preferred juice bases are selected from the following group:
  • Said health drink may also be water-based, such as a mineral water-based product, such as flavoured mineral water-based products.
  • Said flavouring is preferably from fruit juices and/or other natural products.
  • said health drink is an energy shot comprising sugars and other energy-providing products, such as comprised in an 25 or 30 cl bottle.
  • said health drink is an alcoholic beverage, such as a dairy-based alcoholic beverage.
  • said health drink is a meal replacement drinks. It is envisaged that the health drink of the present invention may also be manufactured as a concentrate or premix, ready for making up to the drink at a later stage, preferably by the consumer.
  • the functional food is a solid functional food, such as selected from the group consisting of: Biscuits/crackers, breakfast cereal, soup, muesli, Chewing gum, Sweets (such as boiled sweets), fresh bakery products (fresh bread, cakes, muffins, waffles etc.), dry bakery products (crispbread, biscuits, crackers etc.), cereal products (breakfast cereals, fibre and sterol enriched flours, mueslis, cereal based and muesli bars, such bars possibly containing chocolate, pasta products, snacks etc.), bran products (granulated and/or toasted bran products, flavoured and/or sterol coated bran products and bran-bran mixes etc.).
  • Biscuits/crackers such as selected from the group consisting of: Biscuits/crackers, breakfast cereal, soup, muesli, Chewing gum, Sweets (such as boiled sweets), fresh bakery products (fresh bread, cakes, muffins, waffles etc.), dry bakery products (crispbread
  • said solid functional food is a ready mix (preferably in powder form), either for baking (e.g. breads, cakes, muffins, waffles, pizzas, pancakes) or for cooking (e.g. soups, sauces, desserts, puddings) to be used in preparing or manufacturing of foods
  • baking e.g. breads, cakes, muffins, waffles, pizzas, pancakes
  • cooking e.g. soups, sauces, desserts, puddings
  • said solid functional food is a meat product (sausages, meat-balls, cold cuts etc.)
  • said solid functional food is a bread or morning product/bakery snack.
  • said bread may be white, brown or wholemeal bread.
  • said bread may be selected from the following bread types: malted wheats, milk breads, bran-enriched and mixed grain breads.
  • the bread may be any shape, such as e.g. cob, coburg, cottage, cholla, bloomer, barrel, batch, sandwich, tin, Vienna or farmhouse.
  • said bread is selected from any of the following bread types:
  • Softgrain bread made from white flour with additional grains of softened rye and wheat to increase the fibre content (preferably by 30%) compared with conventional white bread)
  • said bread is selected from any of the following bread types:
  • Soda Bread or brown soda bread (made using wholemeal flour) rye breads baguette or French stick croissants bagel
  • said bread is a flat bread, such as selected from any of the following bread types: Chapattis, Paratas and Roti, Mexican tortilla, flat "wrap” or flour tortilla, pancakes.
  • the functional food is a morning snack or bakery product.
  • Said bakery product may be either sweet or savoury, for example savoury.
  • Preferred bakery products include, but are not restricted to: rolls and baps, toasting products such as muffins, crumpets and pikelets, scones, teacakes, buns and other fruited products, hot plate products such as pancakes and griddle scones, waffles and potato cakes, hot cross buns, croissants, brioches, pain-au-chocolat, bagels, American sweet muffins and other semi-sweet bread products.
  • toasting products such as muffins, crumpets and pikelets
  • scones teacakes
  • hot plate products such as pancakes and griddle scones, waffles and potato cakes
  • hot cross buns croissants, brioches, pain-au-chocolat, bagels, American sweet muffins and other semi-sweet bread products.
  • the functional food is a vegetable oil-based product (spreads, salad oils, mayonnaise etc.)
  • the functional food is a frozen confectionary product.
  • frozen confectionery product includes milk containing frozen confections such as ice- cream, frozen yoghurt, sherbet, sorbet, ice milk and frozen custard, water-ices, granitas and frozen fruit purees.
  • the level of solids in the frozen confection is more than 3 wt %, more preferred from 10 to 70 wt %, for example 40 to 70 wt %.
  • Ice-cream will typically comprise 2 to 20 wt % of fat, 0 to 20 wt % of sweeteners, 2 to 20 wt % of non-fat milk components and optional components such as emulsifiers, stabilisers, preservatives, flavouring ingredients, vitamins, minerals, etc, the balance being water.
  • ice-cream will be aerated e.g. to an overrun of 20 to 400 %, more general 40 to 200 % and frozen to a temperature of from -2 to -200 degrees. C, more general -10 to -30 degrees C. Ice-cream normally comprises calcium at a level of about 0.1 wt %.
  • composition according to the present invention may be encapsulated or combined with emulsifiers, detergents or other agents to ensure solubilisation and stabilisation of the substance in the product.
  • the functional food is a meal replacer.
  • Meal replacer drinks are typically based on a liquid base which may for example be thickened by means of gums or fibers and whereto a cocktail of minerals and vitamins are added.
  • the drink can be flavoured to the desired taste e.g. fruit or choco flavour.
  • a typical serving size may be 330 ml or 330 g.
  • the composition according to the present invention may be encapsulated or combined with emulsifiers, detergents or other agents to ensure solubilisation and stabilisation of the substance in the beverage.
  • Meal replacer snacks or bars often comprise a matrix of edible material wherein the composition according to the present invention can be incorporated.
  • the matrix may be fat based (e.g. couverture or chocolate) or may be based on bakery products (bread, dough, cookies etc) or may be based on agglomerated particles (rice, grain, nuts, raisins, fruit particles).
  • a typical size for a snack or meal replacement bar could be 20-200 g, generally from 40 to 100 g.
  • Further ingredients may be added to the product e.g. flavouring materials, vitamins, minerals etc.
  • the functional food comprises a composition according to the present invention in combination with another survival enhancing agent, longevity enhancing agent, health enhancing agent and/or a modulator of a microbial population.
  • one preferred embodiment of said functional food is a food comprising one or more of the compositions according to the present invention and a probiotic, such as in a probiotic "shot".
  • Another preferred embodiment of the functional food is a food comprising the compounds according to the present invention and a prebiotic, such as in a prebiotic "shot”.
  • Another preferred embodiment of the functional food is a food comprising the compositions according to the present invention and a symbiotic, such as in a symbiotic "shot”.
  • preferred bacteria for use in the above- mentioned shots are any of the following: Lactobacillus sp., such as L. acidophilus, L. casei, L. fermentum, L. johnsonii, L.
  • preferred bacteria for use in the above-mentioned shots are any of the following: Bifidobacterium sp., such as B. bifidium, B. breve, B. lactis, and/or B. longum.
  • preferred bacteria for use in the above-mentioned shots are any of the following: Enterococcus faecalis. Escherichia coli, Saccharomyces boulardii, Saccharomyces cerevisiae and/or Streptococcus thermophilus.
  • composition according to the present invention may be also combined with other ingredients in a dietary supplement, such as e.g. botanical supplements and/or in a vitamin E capsules, or in a selenium pill. Further preferred combination in said dietary supplements may be with e.g. one or more of the following: antioxidant(s), vitamin C, vitamin E, beta-carotene
  • the functional food of the invention can further encompass other healthy components such as for example vitamins A, B, C, D, E, minerals such as calcium, - potassium, magnesium, iron, copper, zinc, selenium and anti-oxidants such as tocopherols, polyphenols.
  • the functional food may comprise a composition according to the invention (such as lentinan) together with vitamin C, the combination capable of causing a reduction in colds and flu in the individual ingesting said functional food.
  • compositions of the invention may comprise further ingredients which are believed to reduce or prevent osteoporosis.
  • ingredients which are believed to reduce or prevent osteoporosis. Examples of such ingredients are calcium, vitamin D, magnesium etc.
  • Beverage comprising any of the compositions described herein in an amount of 0.1- 5%, preferably 0.5-1%.
  • Fresh bakery product comprising any of the compositions described herein in an amount of 0.9-16%, preferably 2.4-10%, and more preferably 3-5%.
  • Dry bakery product comprising any of the compositions described herein in an amount of 1.0-20%, preferably 3.2-15% and more preferably 4.4-10%
  • Cereal product comprising any of the compositions described herein in an amount of 0.8-20%, preferably 1.6-16%, more preferably 2-10%
  • Bran product comprising any of the compositions described herein in an amount of 4%-25%, preferably 6-20% Dairy or non-dairy product (e.g. fermentated cereal product) comprising any of the compositions described herein in an amount of 0.1-20%, preferably 0.8-8%
  • Vegetable oil based product comprising any of the compositions described herein in an amount of 0.6-16%, preferably 2.6-10%, more preferably 2.6-5%
  • Meat product comprising any of the compositions described herein in an amount of 0.1-16%, preferably 0.2-5%.
  • Dairy product comprising: any of the compositions described herein in an amount of 0.1-16%, preferably 0.2-5%.
  • the present invention is concerned with use of any of the compositions described herein in the manufacture of a functional food, such as any of the functional foods described herein.
  • Lentinus edodes was cultivated in liquid culture in medium comprising 15 g/l glucose, 3g/l malt extract, 3 g/l yeast extract and 5 g/l peptone in shake flasks at 25°C. After cultivation the biomass was separated from the rest of the fermentation broth by filtration. The supernatant was subjected to ultrafiltration using a membrane with a nominal pore size of 50,000 Da. The retentate (herein designated the "MediMush product”) was used for analysis of monosaccharide content. For comparison, commercially available Lentinan for injection (Eureka Bio- Chemicals Pty, Little Collins Street. Melbourne 3000, Australia) was used in a reference experiment performed in parallel (herein after this product is designated "Eureka").
  • Samples were hydrolysed to their constituent monosaccharides in 1 N HCI at 100 0 C.
  • the hydrolysates were analysed by HPLC using a Dionex MA-1 column with 60OmM NaOH as the mobile phase; detection was by pulsed amperometry.
  • the major monosaccharides of the MediMush product was mannose and glucose and some galactose.
  • Table A displays the relative monosaccharide content of the MediMush product and Eureka.
  • Soluble protein in the samples was measured by a microscale version of the Coomassie dye-binding assay (Mousdale, D. M, Campbell, M.S., Coggins, J. R. "Purification and characterisation of bifunctional dehydroquinase-shikimate:NADP dehydrogenase from pea seedlings". Phytochemistry 367, 217-222 (1987). The amounts of soluble protein in the two products are shown below:
  • P388 cells are mouse macrophage like cells, which secrete large amounts of interleukin-1 (IL-1) when stimulated (detailed information on the cells is available from the ECACC).
  • the amount of IL-1 secreted can be quantified using an ELISA (Enzyme-Linked ImmunoabSorbant Assay) based method and used as a measure of how stimulated the cells are. It should be noted that no absolute value exist to calibrate such an assay and it is therefore preferred that a reference sample is analysed in parallel and the IL1 production is evaluated as compared to the IL1 production induced by the reference sample.
  • the P388 cells are seeded in a 12 well culture plate and allowed to grow overnight. The various stimulants are added at differing concentrations and incubated at 37 0 C for 24 hr.
  • ELISA Enzyme-Linked ImmunoabSorbant Assay
  • IL-1 antigen
  • the IL-1 is detected using two antibodies against the two different forms of IL-1 , which are raised in a goat (primary antibodies).
  • the next step uses another antibody, which detects the goat antibody (secondary antibody).
  • the antibody is conjugated to horse- radish peroxidase, which gives a colour reaction when substrate is added.
  • the amount of colour measured by a spectrophotometer (ELISA reader) correlates with the amount of IL-1 present thus quantifying how stimulated the tissue culture cells are.
  • the MediMush product was markedly immunostimulatory.
  • the Eureka product showed some immunostimulation but was significantly weaker than the MediMush product.
  • the MediMush product induced production of 58 times more IL1 ⁇ and 20 times more IL1- ⁇ than Eureka.
  • the immune stimulating activity of the MediMush product prepared as described in example 1 was compared to the immune stimulating activity of a polysaccharide composition, which had not been subjected to size fractionation.
  • This product was prepared by cultivating Lentinus edodes in liquid culture as described in example 1. To the supernatant thus obtained around 2 volumes of absolute ethanol was added to precipitate the product. The precipitate was removed, washed with absolute ethanol and re-suspended in distilled water.
  • the immune stimulation assay was performed as described in example 4.
  • the results using the MediMush product is given in table 5 and the results using the precipitated product are given in table 6.
  • the following method may be used: 12 weeks old Sprague Dawley rats receives 1 mg of the composition according to the invention in 0.5 ml 0.09 saline (i.p.) 2 days before the immunisation. Control animals receives 1 mg casein. The animals are immunised with BSA (0.5 mg) in 0.25 "Freunds Complete Adjuvant" and blood samples are obtained after 11 days for measurement of the antibody response. The specific anti-BSA antibody concentration is determined against an absolute standard of antibody BSA by means of "sandwich" ELISA.
  • Biomass Remove the biomass from the broth using a nylon cloth with pore size 45 as a filter medium. Wash the biomass thoroughly with water and dry in a microwave oven set at defrost until dry (normal sample size will require about 15 mins). Store in a desiccator until cool and weigh.
  • Fermentation liquor The concentration of immunostimulating agent in the fermentation liquor is determined by ppt with abs ethanol. Sterile, distilled water is added if necessary to adjust the concentration to the desired level. The resulting liquid is autoclave and stored.
  • Medical grade Pass the biomass-free fermentation liquor through a UF filter having a mwt cut-off such as for example 300 kD. When 70-80% of the liquid has been removed add water to the retentate to wash the solution. Repeat until the solution has lost much (most of) its colour and appears clean.
  • Example 7b Agaricus blazei was cultivated as described in Example 7a, in liquid culture. After cultivation the biomass was separated from the rest of the fermentation broth by filtration. The supernatant was subjected to centrifugal filtration using filters with different mwt cut offs. The first product (denoted MediMush product II) was obtained by removing all components below 10OkD with the remaining liquid containing the product MediMush II. The second product (denoted MediMush product III) was obtained by removing all components from the supernatant having a mwt below 1kD with the remaining liquid containing "Medimush product III ".
  • Samples were hydrolysed to their constituent monosaccharides in 1N HCI at 100 0 C.
  • the hydrolysates were analysed by HPLC using a Dionex MA-1 column with 60OmM NaOH as the mobile phase; detection was by pulsed amperometry.
  • the major monosaccharides of the MediMush products Il and III were mannose and glucose and some galactose.
  • Table B displays the relative monosaccharide content of the MediMush products Il and III .
  • Ganoderma lucidum was cultivated in a similar way as the Agaricus fungus as described in Example 7a, in liquid culture. After cultivation the biomass was separated from the rest of the fermentation broth by filtration. The supernatant was subjected to centrifugal filtration, as illustrated in Example 7a.
  • the first product (denoted MediMush product IV ) was obtained by removing all components below 10OkD with the remaining liquid containing the product MediMush IV.
  • the second product (denoted MediMush product V) was obtained by removing all components from the supernatant having a mwt below 1 kD with the remaining liquid containing "Medimush product V ".
  • mice Twenty-four, 5 week-old, male mice were used in these studies. They were purchased from the Division of Laboratory Animal Medicine, Louisiana State
  • Medimush Product treatment Mice were divided into four groups. Group 1 received 2 mg/kg lentinian in 0.15 M NaCI, intraperitoneal ⁇ (i.p.), group 2 received an equal volume of saline i.p., group 3 received 10 mg/kg Medimush Product in water by gastic gavage (oral) and group 4 received an equal volume of water orally. All mice received treatment once daily for 5 days. Twenty four hours after the last treatment the mice were bled and euthanized for cell isolation. Isolation and stimulation of peritoneal exudates cells (PEC): PEC were collected by peritoneal lavage as previously described (1).
  • PEC peritoneal exudates cells
  • Cells were concentrated to 1 x 10 6 / mL in RPMI 1640 supplemented with 2 mM L-glutamine, 100 units/mL penicillin and 100 ⁇ g/mL streptomycin (medium) and distributed to 24 well tissue culture plates at 1 mL/well. Plates were centrifuged at 1200 x g for 10 min and placed at 37 0 C, 5%CO 2 for 2 hours. Medium was discarded and the wells washed once with warm medium and replaced with 1.5 ml. warm medium supplemented with 10% fetal bovine serum (Hyclone, Inc. Logan, UT) (complete medium). Half the wells from each group were treated with E.
  • LPS coli lipopolysaccharide
  • the phenotype of the cells was determined by flow cytometry using antibodies to CD3 and CD14 (eBioscience, San Diego, CA) labeled with phycoerythrin and anti-mouse IgG labeled with fluorescein (Jackson ImmunoResearch, Avondale, PA).
  • Isolation and stimulation of splenocytes Spleens were harvested from each mouse. A portion was snap frozen for future mRNA analysis.
  • the remaining spleens were pooled by group and crushed through a 70 ⁇ m nylon screen (Becton Dickenson Labware, Franklin Lakes, NJ). Splenocytes were washed once and the pellet resuspended in buffered ammonium chloride for 5 min on ice. Cells were washed twice and resuspended at 4 x 10 7 VmL in complete medium distributed to 24 well tissue culture plates at 1.5 mL/well. Representative wells were stimulated with E. coli LPS at 10 ng/ml or Concanavilin A (ConA; Sigma Chemical Co.) at 5 ⁇ g/mL and the cells were placed at 37 0 C, 5%CO 2 for 12 or 24 hours. ELISA for cytokines.
  • Cytokine ELISA for TNF ⁇ , IL-1 ⁇ , IL-6 and IL-12 were conducted on serum and culture supernatants exactly as described by the manufacturer (eBioscience). Undiluted supernatants were tested in triplicate. Serum was diluted 1 : 10 for testing.
  • Medimush Product had no statistically significant effect on their growth as measured by weight gain (Table 1). However, i.p. Medimush Product had an effect on the number and phenotype of the cells isolated from the peritoneum lavage. Orally exposed mice and those that received saline i.p were relatively similar; however, there was a increase in the total number of cells isolated from the peritoneum of i.p. Medimush Product treated mice (data not shown). The increased number of cells was due mainly to an increase in neutrophils in the population (Table 2). The relative contribution to the observed results by the PMN compared to macrophage cannot be estimated at this time, but might effect the difference observed differences in the i.p. groups.
  • IL-1 ⁇ production by PEC (18-fold and 6-fold, respectively; Table 4).
  • IL-1 ⁇ in response to in vitro LPS was also increased more than 3-fold in ip treated mice
  • IL-1 ⁇ production by PEC from mice exposed to oral Medimush Product and stimulated in vitro LPS was decreased (2-fold).
  • Splenocytes from treated and untreated mice produce small amounts of IL-1 ⁇ in the first 12 hours of culture.
  • splenocytes from the mice were stimulated to produce IL-1 ⁇ by treatment with either LPS or ConA (a T cell mitogen).
  • Oral Medimush Product exposure generally decreased pro- inflammatory cytokine production by PEC and increased pro-inflammatory production by splenocytes suggesting the systemic response to Medimush Product (oral) may be different from the local responses measured in previous studies (nasal, i.p., i.v.).
  • intraperitoneal control 6 14 17 46 18 intraperitoneal Medimush 4 3 84 6
  • Medimush Product P c intraperitoneal none 1 + 1 5 + 1** 0.0084 intraperitoneal LPS 2 + 0 49 + 9** 0.0015 intraperitoneal Con A 4 + 2 13 + 3 0.0717 oral none 3+ 1 3 + 0 0.7811 oral LPS 3 + 0 11 + 1** 0.0213 oral ConA 3 + 1 11 + 2 0.0969 a.
  • Medimush Product P c intraperitoneal none 567 + 25 320 + 33** 0.0194 intraperitoneal LPS 265 + 15 286 + 54 0.5657 oral non 288 + 30 247 + 30** 0.0027 oral LPS 258 + 31 209 + 17 0.8763 a.
  • Medimush Product P c intraperitoneal none 132 + 2 344 + 30** 0.0098 intraperitoneal LPS 299+ 10 367 + 31 _ 0.0853 intraperitoneal Con A 393 + 19 233 + 24** 0.0029 oral none 114 + 2 199 + 7** 0.0014 oral LPS 367 + 31 329+ 11** 0.0124 oral ConA 233 + 24 248 + 25** 0.0284 a.
  • Medimush Product P c intraperitoneal none 101 + 2 272 + 5** 0.0009 intraperitoneal LPS 624 + 15 932 + 17 0.0011 oral none 764 + 10 852 + 43 0.0833 oral LPS 835 + 29 760 + 77 0..3680 a.
  • Limit of detection 8 pg/ml b. 0.15 M saline was used as a control for intraperitoneal injections and distilled water was used as a control for oral exposure.
  • Medimush Product P c intraperitoneal none 105 + 6 638 + 18** 0.0004 intraperitoneal LPS 218+ 16 524 + 33** 0.0042 intraperitoneal Con A 567 + 7 835 + 39** 0.0029 oral none 117 + 5 190 + 3** 0.0007 oral LPS 174 + 5 266+ 3** 0.0065 oral ConA 326 + 6 510 + 11** 0.0039 a.
  • Medimush Product P c intraperitoneal none 1 + 1 11 + 5 0.1017 intraperitoneal LPS 13 + 9 2 + 1 0.2611 intraperitoneal Con A 96 + 11 86 + 8 0.4155 oral none 2 + 2 0 + 0 0.3855 oral LPS 4 + 4 1 + 1 0.4405 oral ConA 71 + 11 80 + 7 0.2293 a.
  • Example 10 Bar 75 g of dark chocolate are melted at 70 degrees C and subsequently mixed with 600 mg of Medimush product. The mixture is poured into a bar shaped mold and cooled overnight.
  • vanilla flavoured ice-cream 100 ml of vanilla flavoured ice-cream are mixed with 100 ml of cooled milk, 10 ml of strawberry syrup and 1 ml extracellular liquid from mushroom cultivation. The mixture is fed through a blender and immediately served.
  • Method of preparation dissolve hyfoama and gelatin in water add 150 g of sugar and beat to foam, heat remaining sugar to 130 degrees C and add slowly to foam. Add fat, glucose syrup, milkpowder and 1 g extracellular liquid from mushroom cultivation. Allow to cool and divide in bars of 50 g.
  • Example 13 Fruit Drink

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Abstract

L'invention se rapporte à des compositions contenant des polypeptides et des polysaccharides. Ces compositions sont généralement à modulation immunitaire. Cette invention se rapporte aussi à des procédés de fabrication de ces compositions au moyen de champignons filamenteux cultivés dans un milieu liquide. Ces compositions sont utiles par exemple dans le traitement de conditions de faiblesse immunitaire.
EP05758000A 2004-07-16 2005-07-15 Composes a modulation immunitaire issus de champignons Withdrawn EP1774012A2 (fr)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
US10/892,393 US7514085B2 (en) 2004-07-16 2004-07-16 Immune modulating compounds from fungi
US69047705P 2005-06-15 2005-06-15
US69049605P 2005-06-15 2005-06-15
US69048205P 2005-06-15 2005-06-15
DKPA200500882 2005-06-15
DKPA200500881 2005-06-15
DKPA200500880 2005-06-15
PCT/DK2005/000498 WO2006007848A2 (fr) 2004-07-16 2005-07-15 Composes a modulation immunitaire issus de champignons

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