Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
  • G01N33/6869—Interleukin
• • 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
  • A61K39/02—Bacterial antigens
  • A61K39/07—Bacillus
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
• • 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
  • A61K39/35—Allergens
• • 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
  • A61P11/02—Nasal agents, e.g. decongestants
• • 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
  • A61P11/06—Antiasthmatics
• • 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
  • A61P37/08—Antiallergic agents
• • 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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6854—Immunoglobulins
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
  • G01N33/6866—Interferon
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2400/00—Lactic or propionic acid bacteria
  • A23V2400/11—Lactobacillus
  • A23V2400/113—Acidophilus
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2400/00—Lactic or propionic acid bacteria
  • A23V2400/11—Lactobacillus
  • A23V2400/125—Casei
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
  • G01N2333/335—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Lactobacillus (G)

Definitions

• the invention relates to the field of allergic disease prevention and/or treatment, and in particular to probiotic bacteria which have the capacity to prevent and/or treat allergic disease.
• BACKGROUND ART Allergy is a clinical syndrome affecting about one third of the population, manifest as rhinitis, asthma, eczema or food hypersensitivity.
• target tissue changes play a role in determining the pattern of disease
• the central abnormality (known as atopy) is the genetically influenced propensity to develop an IgE antibody response following antigen exposure.
• atopy the central abnormality
• the characteristics of the gut bacterial flora may drive IgE-promoting immunological mechanisms possibly by affecting the cytokine balance produced by CD4+ T lymphocytes.
• the present invention provides a method of lowering IgE levels by administration of a therapeutically effective amount of live probiotic bacteria, or a live probiotic bacteria-containing composition, to a subject in need thereof.
• a method of prophylactic or therapeutic treatment of allergy by administration to a subject requiring such treatment a therapeutically effective amount of live probiotic bacteria, or a live probiotic bacteria- containing composition preferably, the probiotic bacteria is, or the probiotic bacteria-containing composition includes. Lactobacillus.. Most preferably, the Lactobacillus is Lactobacill s acidophil s and/or Lactobacillus casei.
• IgE is lowered from elevated levels induced by an allergen or as a consequence of an allergic disorder.
• the probiotic bacteria, or a probiotic bacteria-containing composition is administered at the time of exposure to an allergen or shortly thereafter.
• the term "exposure" with reference to an antigen or allergen includes natural exposure such as for example day-to-day contact with or ingestion of food products and the like, or seasonal exposure or re-exposure to allergens such as pollen or other air-borne allergens or by contact with skin and other body surfaces which may involve contact with synthetic materials or natural substances.
• allergens such as pollen or other air-borne allergens or by contact with skin and other body surfaces which may involve contact with synthetic materials or natural substances.
• the probiotic bacteria or compositions containing such bacteria are preferably administered at the time of exposure to the antigen/allergen or shortly thereafter.
• the treatment is preferably commenced at the beginning of the season or shortly thereafter.
• the term “exposure” includes natural exposure such as for example day-to-day contact with or ingestion of food products and the like, or seasonal exposure or re-exposure to allergens such as pollen or other air-borne allergens or by contact with skin and other body surfaces which may involve contact with synthetic materials or natural substances.
• antigen/allergen when used with reference to antigen/allergen also includes artificial exposure or administration such as for example the injection of antigen/allergen in desensitisation procedures or under-tongue administration of antigen/allergen.
• the antigen/allergen is preferably co-administered (co-presented) with the probiotic bacteria or a composition containing them.
• probiotic bacteria may also be administered shortly after administration of the antigen/allergen.
• the subject in need thereof is selected from the group consisting of high risk infants; those subjected to high risk occupational exposure to allergens; those exposed to high risk allergens; those having recognised allergy to specific allergens; and those prone to anaphylaxis.
• the high risk infants are children of parents who both have allergic disease.
• those subject to high risk occupation exposure to allergens are selected from the group consisting of aluminium smelter workers, woodworkers, chemical factory workers, and those working with latex-containing materials, especially gloves.
• those exposed to high risk allergens are exposed to bee venom.
• exposure to high risk allergens is parenteral exposure.
• the specific allergens are present in the pre-pollen season, foods or latex-containing materials.
• the trigger for the allergic response is insect envenomation, and food and drug sensitivities.
• the probiotic bacteria or probiotic bacteria-containing composition is in tablet or capsule form.
• the probiotic bacteria may be present in a food source such as a yoghurt or other dairy product.
• the amount of probiotic bacteria administered to a human subject is at least 10 10 live bacteria. More preferably the amount administered is from about 10 10 to about 10 n live bacteria.
• the required dosage amount will vary according to the severity of the allergic condition, the nature of the allergic condition, age of the subject and other standard clinical parameters. These parameters as well as the required dosage can be easily assessed by those skilled in the art. In human subjects it is preferred that the probiotic bacteria or a composition containing them be administered daily.
• the present invention provides the use of live probiotic bacteria for the manufacture of a medicament for lowering IgE levels.
• the present invention provides use of live probiotic bacteria for the manufacture of a medicament for treating allergy.
• the present invention provides a method of identifying a bacterial species capable of lowering IgE levels in a mammal including: a) administration of the bacterial species to a mammal; b) administration of an allergen (antigen) to the mammal; and c) determination of the IgE antibody level in the mammal after treatment with the bacterial species and comparison with a control mammalian which a bacterial species
• steps (a) and (b) were not administered, wherein steps (a) and (b) can be performed consecutively in any order or simultaneously.
• the bacterial species is a Lactobacillus species and most preferably it is Lactobacillus acidophilus or Lactobacillus casei.
• the animal model for identifying useful probiotic bacteria preferably makes use of the mouse.
• other animal models may be developed on the same principle as disclosed herein.
• the bacterial species is preferably administered orally however it may also be administered intraperitoneally and other means. Most preferably, the
• 15 bacterial species is administered in an amount of 10 8 to 10" bacteria, and more preferably, in an amount of 0.6 to 1.0 x 10'° bacteria.
• 1 to 20 oral doses are administered prior to administration of the antigen and most preferably 4 to 8 oral doses are administered prior to administration of the antigen.
• administration of the oral doses is at 1 to 5 day intervals and, most preferably, at 2 day intervals.
• 0 administration is over a 1 to 3 week period and, most preferably, over a 1 to 2 week period.
• the antigen is administered at 5 a dose of 4 to 10 ⁇ g, and most preferably, at a dose of 8 ⁇ g.
• the antigen is administered intraperitoneally.
• the bacterial species is administered at the same time or 1 day after administration of the antigen/allergen.
• the antigen/allergen can be administered after the bacterial species and in such circumstances the antigen/allergen is administered preferably 1 day after the 0 administration of 4 to 8 oral doses of bacteria.
• administration of the bacteria species is continued after administration of the antigen. Most preferably, approximately 8 oral doses of bacteria are administered.
• the bacteria are administered at 2 day intervals.
• the IgE antibody is obtained from serum.
• the serum is collected approximately 14 days after administration of the antigen.
• the IgE antibody level is determined using an ELISA assay.
• the skilled addressee will recognise that other IgE antibody assays may also be used.
• the allergic disease is selected from the group consisting of asthma, eczema, hayfever and food allergy.
• the present invention provides a bacterial species identified by the method of the fourth aspect.
• the present invention provides a composition including a bacterial species according to the fourth aspect.
• composition is in the form of a capsule or tablet or similar formulation however it may also be in the form of a food product.
• a pharmaceutical composition including an effective amount of live bacterial species according to the fourth aspect, together with a pharmaceutically acceptable carrier, adjuvant, solvent or excipient.
• the bacterial species is L. acidophilus.
• a method of assessing efficacy of treatment with live probiotic bacteria or with a composition having live probiotic bacteria including the steps of: a measuring the level of salivary immunoglobulin subclass in a sample obtained before commencement of treatment, b measuring the level of salivary immunoglobulin subclass in a sample obtained after commencement of treatment c comparing the levels of salivary immunoglobulin subclass in a) and b), wherein the change in immunoglobulin subclass level is indicative of effective treatment.
• the immunoglobulin subclass is IgGl or IgG2 and it will be understood that equivalent subclasses in various species can also be advantageously used.
• a method of assessing efficacy of treatment with live probiotic bacteria or with a composition having live probiotic bacteria including the steps of: a measuring the level of LL-4 or IFN- ⁇ in a sample obtained before commencement of treatment, b measuring the level of IL-4 or IFN- ⁇ in a sample obtained after commencement of treatment c comparing the levels of IL-4 or IFN- ⁇ in a) and b), wherein the change in IL-4 or
• IFN- ⁇ level is indicative of effective treatment. It will be clear however that any know cytokine marker for the Thl or the Th2 response will be suitable in place of IFN- ⁇ or IL-4.
• Figure 1 Suppression of IgE antibody response in mice fed probiotic bacteria.
• Figure 5 Suppression of IgE response to OVA, and cytokine and IgG antibody subclass response, by L acidophilus
• a mouse animal model was developed which enabled identification of probiotic bacteria capable of downregulating the IgE response to an antigen/allergen.
• the animal used is a mouse (for example C57BL/6).
• the animals are fed the candidate bacterial species (alive, dose 10 8 -10" , frequency every 1 -5 days, for 1 -3 weeks).
• the animals are fed the bacteria at the same time as the antigen was being introduced or shortly thereafter, for example the following day.
• Mice were immunised intraperitoneally with antigen (for example ovalbumin) at an appropriate dose (eg 4-1 O ⁇ g, preferably 8 ⁇ g), with specific and total IgE measured in serum (eg. at 14 days after immunisation).
• antigen for example ovalbumin
• specific and total IgE measured in serum eg. at 14 days after immunisation.
• feeding with test bacteria continues throughout the experiment (see examples).
• comparisons are made with killed organisms and dose-response is estimated.
• timing of administration of probiotic bacteria in relation to antigen/allergen challenge is examined. The examples demonstrate: (i) that frequent dosage with live probiotic bacteria at an appropriate does can reduce the IgE response to antigen/allergen.
• oral administration of the bacterial species affects the systemic IgE antibody response (following injected antigen) and thus may be of value in subjects who have anaphylaxis (eg insect envenomation, and food and drug sensitivities).
• probiotic bacteria can downregulate IgE production following antigen administration or exposure to antigen - thus continuous oral ingestion of appropriate doses of live bacteria selected by this method may be of value in the ongoing management of allergic disease.
• administration of live probiotic bacteria is particularly effective
• co-administration of probiotic bacteria with the antigen/allergen, or administration of probiotic bacteria shortly after the antigen/allergen challenge is particularly effective
• the probiotic bacteria can be formulated into various compositions and preferably the compositions are pharmaceutical compositions in the form of capsules, tablets, powders and the like.
• Such formulations can be prepared by known means, using pharmaceutically acceptable carriers, excipients, solvents or adjuvants. Such procedures and ingredients are well know and amply described in standard texts and manuals, for example "Remington: The Science and Practice of Pharmacy", 1995, Mack Publishing Co. Easton, PA 18042, USA, which is incorporated herein by reference.
• the probiotic bacteria may also be formulated into food products by the usual well known means.
• Bacteria ⁇ Lactobacillus acidophilus or Lactobacillus casei) were grown in MRS agar petri dishes (3.8% w/v, Oxoid, Basingstoke, UK). Plates were incubated in 5% CO 2 and air in a humid atmosphere at 37°C for 48 hours.
• PBS phosphate buffered saline
• mice Thirty female C57/B16 SPF mice (Animal Resource Centre, Perth, WA, Australia) were fed with 4 or 8 oral doses of 0.6-1.0 x 10 10 L acidophilus or L casei in 0.2 mL PBS or PBS alone given two days apart.
• mice were injected intraperitoneally with 7 ⁇ g of egg albumin (ovalbumin. OVA, Sigma-Aldrich, St Loius, Missouri, USA) and 4 mg aluminium hydroxide (Amphojel, Whitehall Laboratories, Sydney, Australia) in 0.2 mL PBS or with PBS alone.
• egg albumin ovalbumin. OVA, Sigma-Aldrich, St Loius, Missouri, USA
• aluminium hydroxide aluminium hydroxide
• mice Immunised and control mice were then placed on a feeding regime consisting of 8 oral doses of 0.6-1.0 x 10'° bacteria in PBS or PBS alone given two days apart. Mice were bled by the saphenous vein 2 days after the last dose and the serum collected for IgE antibody determination using an ELISA assay.
• Example 2 Suppression of IgE response to OVA by L acidophilus after allergen sensitisation
• mice were fed 10 10 L acidophilus before or 24 hrs after sensitisation with 8 ⁇ g ovalbumin (OVA) in alum per mouse.
• Control mice were sham fed with normal saline.
• a total of 10 feeds was administered before they were assessed for levels of OVA-specific IgE antibody and total IgE in serum.
• L acidophilus was more effective if administered (or co-administered) at the time of exposure to OVA than when administered prior to allergen exposure, as shown by the suppression of OVA-specific IgE antibody and total IgE responses (Figure 2 ).
• Data are mean + SEM from 5-10 mice. *, p ⁇ 0.05 compared with control values from saline-fed sensitised mice.
• Example 3 Suppression of IgE response to OVA by L. acidophilus is dose- dependent Mice were fed orally with 10 8 . 10 9 or 10 10 L acidophilus before they were sensitised 24 hrs later with OVA in alum. Control mice were sham fed PBS. Each dose was administered 10 times every 2 days for 21 days. One week following the final dose, serum IgE and OVA-specific IgE antibody were measured. A dose-dependent suppression of IgE and OVA-specific IgE antibody was noted with a statistically significant effect with 10 l ⁇ bacteria ( Figure 3). Data are mean + SEM from 10 mice. *, p ⁇ 0.05 compared with control values from saline-fed sensitised mice.
• mice were fed live or formalin-killed 10 10 L acidophilus and then sensitised with OVA as per standard protocol described in Example 3.
• Figure 4 shows that live bacteria were more effective in suppressing IgE response than killed bacteria. Data are mean + SEM from 5 mice. *, p ⁇ 0.05 compared with values from saline-fed sensitised mice.
• Example 5 Suppression of IgE response to OVA by L acidophilus correlates with cytokine and IgG antibody subclass response
• mice fed 10'° L acidophilus produced higher amounts of IFN- ⁇ and lower amounts of IL-4 in the spleen, a finding consistent with the suppression of IgE response.
• the contrasting cytokine patterns correlated with the production of salivary IgG subclass antibodies which showed an upregulation of IgG2a antibody and a downregulation of IgGl , respectively ( Figure 5 ).
• Data are mean + SEM from 10 mice. *, p ⁇ 0.05; **, p ⁇ 0.01 compared with values from saline-fed sensitised mice.

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