US20050214270A1 - Antiallergic agent, utilization thereof for reducing allergy and method of reducing allergy - Google Patents

Antiallergic agent, utilization thereof for reducing allergy and method of reducing allergy Download PDF

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US20050214270A1
US20050214270A1 US10/518,801 US51880105A US2005214270A1 US 20050214270 A1 US20050214270 A1 US 20050214270A1 US 51880105 A US51880105 A US 51880105A US 2005214270 A1 US2005214270 A1 US 2005214270A1
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lactic acid
acid bacteria
lactobacillus acidophilus
antigen
ferm
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Naouki Yamamoto
Yuu Ishida
Izuki Bando
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Asahi Soft Drinks Co Ltd
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Assigned to CALPIS CO., LTD. reassignment CALPIS CO., LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BANDO, IZUKI, ISHIDA, YUU, YAMAMOTO, NAOYUKI
Publication of US20050214270A1 publication Critical patent/US20050214270A1/en
Priority to US12/253,819 priority Critical patent/US8003092B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • 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
    • 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
    • A61P17/00—Drugs for dermatological disorders
    • 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
    • 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
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/853—Lactobacillus
    • Y10S435/854—Lactobacillus acidophilus

Definitions

  • the present invention relates antiallergic agents.
  • the invention also relates to use of the antiallergic agents for reducing allergy, and a method for reducing allergy.
  • Allergic patients have been increasing in number every year in many countries including Japan, and high incident of allergic adults, one out of three in Japan, is reported. Allergic diseases are categorized into four types, type I to IV, depending on their mechanism of action. Some kinds of allergic rhinitis such as pollinosis, bronchial asthma, and atopic dermatitis are Type I immunoglobulin E (IgE)-mediated allergy, wherein increase in antigen-specific IgE level in blood enhances the risk of developing allergic symptoms.
  • IgE immunoglobulin E
  • Type I allergy The mechanism of development of Type I allergy is as follows.
  • an antigen such as pollens, house dust, or mites
  • an IgE antibody specific to such antigen is produced, and binds to mast cells or Fc ⁇ receptors on the basophil surface to sensitize the subject.
  • the antigen binds to the IgE antibody to form a complex. This causes degranulation, wherein chemical mediators in the granules, such as histamine and leukotoriene, are released to develop allergic symptoms.
  • antiallergic agents that are easy to take and highly safe are desired. Accordingly, novel antiallergic agents having such properties are demanded.
  • the present inventors have constructed a mouse model wherein the antigen-specific IgE level has remarkably been elevated without substantial increase in the IgG level. Using this model, the inventors have made researches on IgE-level repressing effect of various lactic acid bacterial strains that may affect the intestinal immune system, to find out that, among the various tested lactic acid bacteria, certain bacteria have a particularly excellent inhibitory effect on IgE production, thereby completing the present invention.
  • an antiallergic agent comprising, as an active ingredient, lactic acid bacteria selected from the group consisting of lactic acid bacteria of the species Lactobacillus acidophilus , lactic acid bacteria of the species Lactobacillus fermentum , and combinations thereof.
  • said lactic acid bacteria of the species Lactobacillus acidophilus are bacteria of the strain selected from the group consisting of Lactobacillus acidophilus CL0062 (deposited at International Patent Organism Depositary, FERM BP-4980), Lactobacillus acidophilus CL92 (deposited at International Patent Organism Depositary, FERM BP-4981), and combinations thereof.
  • said lactic acid bacteria of the species Lactobacillus fermentum are of the strain Lactobacillus fermentum CP34 (deposited at International Patent Organism Depositary, FERM BP-8383).
  • the antiallergic agent mentioned above which reduces, when administered orally, antigen-specific IgE level in blood in a mouse rhinitis model wherein antigen-specific IgE level in blood has been elevated by nasally exposing the mouse to continuous antigen stimulation.
  • a method for reducing allergy comprising administering an effective dose of the antiallergic agent mentioned above to a subject in need of such reduction.
  • FIG. 1 shows graphs indicating the changes in immunoglobulin level in blood in IgE-elevated mice in Example 1.
  • FIG. 2 is a graph showing the results of experiments for suppressing OVA-IgE level in IgE-elevated mice by administration of fermented milks, conducted in Example 2.
  • FIG. 3 is a graph showing the results of experiments for suppressing OVA-IgE level in IgE-elevated mice by administration of fermented milks, conducted in Example 3.
  • FIG. 4 is a graph showing the results of experiments for suppressing OVA-IgE level in IgE-elevated mice by administration of fermented milks, conducted in Example 4.
  • FIG. 5 is a graph showing the results of experiments for suppressing allergic symptoms in human by administration of fermented milk, conducted in Example 5.
  • FIG. 6 is a graph showing the results of experiments for suppressing allergic symptoms in human by administration of fermented milk, conducted in Example 5.
  • the antiallergic agent according to the present invention contains, as an active ingredient, lactic acid bacteria selected from the group consisting of lactic acid bacteria of the species Lactobacillus acidophillus , lactic acid bacteria of the species Lactobacillus fermentum , and combinations thereof.
  • the lactic acid bacteria of the species Lactobacillus acidophilus may particularly preferably be of the strain Lactobacillus acidophilus CL0062 (deposited at International Patent Organism Depositary of Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan, under the deposit number FERM BP-4980 on Mar. 4, 1994), the strain Lactobacillus acidophilus CL92 (deposited at International Patent Organism Depositary under the deposit number FERM BP-4981 on Mar. 4, 1994), or a combination of these.
  • the lactic acid bacteria of the species Lactobacillus ferment un may particularly preferably be of the strain Lactobacillus fermentum CP34 (deposited at International Patent Organism Depositary under the deposit number FERM BP-8383 on May 23, 2002).
  • Lactobacillus acidophilus CL0062 strain has the following bacteriological properties:
  • Lactobacillus acidophilus CL92 strain has the following bacterial properties:
  • Lactobacillus fermentum CP34 strain has the following bacteriological properties:
  • the content of the above-mentioned lactic acid bacteria in the antiallergic agent of the present invention is not particularly limited, and may suitably be adjusted depending on ease of production or a preferred daily dosage.
  • a preferred content of the bacteria is from 1 ⁇ 10 7 cells/ml to 1 ⁇ 10 10 cells/ml.
  • the antiallergic agent of the present invention may optionally contain other components, in addition to the lactic acid bacteria.
  • other components may include additives such as excipients, or components of the medium to be discussed later.
  • the antiallergic agent of the present invention may be prepared by culturing the lactic acid bacteria in a medium.
  • Any medium may be used for culturing, as long as the above-mentioned lactic acid bacteria may grow therein, and animal milk, skim milk, milk whey, MRS medium, GAM medium, BL medium, Briggs Liver Broth, or other synthetic media may be used.
  • the temperature for the culture may be 25° C. to 50° C., preferably 35° C. to 42° C.
  • the culture time may be 3 hours to 48 hours, preferably 8 hours to 12 hours.
  • the cultured medium may be used as the antiallergic agent of the present invention with or without further processing.
  • the bacterial cells harvested from the cultured medium by centrifugation or filtration, a lyophilized product thereof, a heat-treated product thereof, or ground bacterial cells may be used as the antiallergic agent of the present invention.
  • the bacterial cells in the above forms may further be formulated, or blended in various food materials such as beverages, tablets, pastes, or bread, before use as the antiallergic agent of the present invention.
  • the antiallergic agent of the present invention may be administered by any route, but oral administration is preferred.
  • the dosage may be not lower than 2 ⁇ 10 9 cells per day, preferably 2 ⁇ 10 10 cells per day for oral administration to human. This dosage of agent may be administered in a single dose or in a plurality of doses per day.
  • the antiallergic agent of the present invention effectively suppresses the IgE level as will be demonstrated in Examples, and is expected to be highly safe since the active ingredient of this agent is bacterial cells taken as food.
  • the method for reducing allergy according to the present invention includes the step of administering an effective dose of the antiallergic agent mentioned above to a subject in need of such reduction.
  • the subject may be animals such as human or other mammals.
  • the antiallergic agent of the present invention effectively suppresses the IgE level in living organisms, and is easy to take and highly safe.
  • the present agent is useful for suppressing allergy involving excess IgE level.
  • mice Male BALB/c mice were obtained from Charles River Japan, and raised under free access to CE-2 (CLEA Japan, Inc.) as a feed. 10 ⁇ g of ovalbumin (abbreviated as OVA hereinbelow, manufactured by SIGMA CHEMICAL CO.) and 2 mg of aluminum hydroxide (WAKO PURE CHEMICAL INDUSTRIES, LTD.) as an adjuvant were suspended in 300 ⁇ l of saline. Ten of the above mice at six weeks old were injected intraperitoneally with this suspension on the first day of sensitization and on day 4 for primary sensitization.
  • OVA ovalbumin
  • the nose of each mouse was soaked in an OVA antigen solution containing 25 mg OVA/ml of saline for three seconds, and this soaking operation was repeated three times as one cycle. Two cycles of soaking operation was performed per day, and the daily soaking was performed from day 10 to day 16 to prepare IgE-elevated mice.
  • the blood OVA-IgE level was measured by the sandwich ELISA. 100 ⁇ l of saline solution containing 10 ⁇ g/ml of a sheep polyclonal anti-mouse IgE antibody (trade name AAM11, manufactured by DAINIPPON PHARMACEUTICAL CO., LTD.) was added to each well of a 96-well immunoplate (manufactured by CORNING INCORPORATED), and incubated overnight at 4° C.
  • a sheep polyclonal anti-mouse IgE antibody trade name AAM11, manufactured by DAINIPPON PHARMACEUTICAL CO., LTD.
  • the plate was washed three times with a phosphate buffer (containing 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2 HPO 4 , and 1.5 mM KH 2 PO 4 , abbreviated as PBS hereinbelow), coated with 0.5% casein-PBS, and incubated for 3 hours at room temperature. After the plate was washed three times with PBS, 100 ⁇ l of a 1/10 PBS dilution of a serum sample was added to each well, and reacted overnight at 4° C.
  • a phosphate buffer containing 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2 HPO 4 , and 1.5 mM KH 2 PO 4 , abbreviated as PBS hereinbelow
  • 0.2 M citric acid buffer prepared by mixing 0.2 M citric acid and 0.2 M trisodium citrate and adjusting the pH to 5
  • ABTS 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
  • 0.006% hydrogen peroxide was added to each well, and shielded for 3 hours at 37° C. for coloration.
  • OD 405 and OD 492 were measured, and the true optical density was obtained by OD 405 value ⁇ OD 492 value.
  • a blood sample was obtained from a mouse that had been injected intraperitoneally with 25 mg/ml of OVA in saline five times (once a week). From the blood sample, a serum sample was prepared as a standard serum. This standard serum was diluted 1/10 with PBS, and the resulting dilution was further diluted stepwise to twice with non-immunized serum to prepare working dilutions. These working dilutions were subjected to measurements of the coloring values in accordance with the above procedures, to obtain a working curve. Based on this working curve, the OVA-IgE levels in the serum samples were obtained as relative amounts with respect to the OVA-IgE level in the standard serum being as 1.
  • saline solution containing 10 ⁇ g/ml of a sheep polyclonal anti-mouse IgE antibody (trade name AAM11, manufactured by DAINIPPON PHARMACEUTICAL CO., LTD.) was added to each well of a 96-well immunoplate (manufactured by CORNING INCORPORATED), and incubated overnight at 4° C.
  • the plate was washed three times with PBS, coated with 0.5% casein-PBS, and incubated for 3 hours at room temperature. After the plate was washed three times with PBS, 50 ⁇ l of a 1/25 dilution of a serum sample in 0.5% casein-PBS was added to each well, and reacted overnight at 4° C.
  • mouse anti-DNP-IgE (manufactured by YAMASA CORPORATION), instead of the serum samples, was dissolved in 0.5% casein-PBS at various concentrations, and subjected to the same procedures as above to obtain a working curve. Based on this working curve, the total IgE levels in the serum samples were calculated.
  • Each of the lactic acid bacterial strains shown in Table 1 was precultured in MRS medium overnight at 37° C., and the cells were harvested by centrifugation at 3000 rpm for 10 minutes.
  • 9% (W/V) reconstituted skim milk (containing 0.1% (W/V) yeast extract (manufactured by DIFCO)) was fermented with the collected cells at 37° C. until the milk was coagulated. After the fermentation, the total cell count of each fermented milk was measured. The results are shown in Table 1.
  • mice were prepared in the same way as in Example 1, and the blood OVA-IgE was measured on day 18 of sensitization in the same way as in Example 1.
  • the mice were divided into groups of 10 mice per group with the same average of blood OVA-IgE levels. From day 19 to 21 of sensitization, various fermented milks shown above, non-fermented 9% (W/V) reconstituted skim milk, or non-fermented 9 w/v % reconstituted skim milk containing 750 ⁇ g of cyclophosphamide were administered gastrically to each group of mice in dosages of 1 ml per day for three days.
  • W/V non-fermented 9%
  • 9 w/v % reconstituted skim milk containing 750 ⁇ g of cyclophosphamide
  • mice On day 22 of sensitization, blood samples from the mice were obtained from the ophthalmic veins, and serum samples were prepared. The blood OVA-IgE and the total IgG levels were measured. As a control, a blood sample from a mouse, which had been sensitized in the same way but given no fermented milk or the like, was obtained in the same way, and the blood OVA-IgE and the total IgG levels were measured. The results are shown in FIG. 2 .
  • Example 2 The procedure in Example 2 was followed except that the lactic acid bacterial strains shown in Table 2 were used. The results of measurement of the total cell count in each fermented milk are shown in Table 2. The results of measurement of the blood OVA-IgE are shown in FIG. 3 .
  • TABLE 2 Total cell count Strain (cells/ml) Lactobacillus acidophilus CL0062 (BP-4980) 4.40 ⁇ 10 8 Lactobacillus gasseri CP2209 4.30 ⁇ 10 8 Lactobacillus reuteri ATCC23272 9.60 ⁇ 10 8 Bifidobacterium breve CP2425 1.30 ⁇ 10 8
  • Lactobacillus acidophilus CL92 strain and Lactobacillus fermentum CP34 strain were respectively precultured in MRS medium overnight at 37° C., and the cells were harvested by centrifugation at 3000 rpm for 10 minutes. The collected cells were cultured in MRS medium overnight at 37° C., and the cells were harvested by centrifugation at 3000 rpm for 10 minutes. The number of cells was measured for each strain, and the cells were suspended in 9% skim milk at a concentration of 1 ⁇ 10 6 cells per 1 ml to obtain suspensions.
  • mice IgE-elevated mice were prepared in the same way as in Example 1, and the blood OVA-IgE was measured on day 18 of sensitization in the same way as in Example 1.
  • the mice were divided into groups of 10 mice per group with the same average of blood OVA-IgE levels. From day 19 to 21 of sensitization, the above suspensions were administered gastrically to each group of mice in dosages of 1 ml per day for three days. On day 22 of sensitization, blood samples from the mice were obtained from the ophthalmic veins, and serum samples were prepared. The blood OVA-IgE and the total IgG levels were measured. The results are shown in FIG. 4 .
  • a represents the standard ratio of OVA-IgE level when the non-fermented skim milk was fed
  • b represents the standard ratio of OVA-IgE when each suspension was fed.
  • Frequency of nose blowing also showed a tendency to decrease three weeks after the commencement of intake (Wilcoxon test: p ⁇ 0.1) During the period of intake, tendency of decrease in frequency of sneezing, remission of swelling of inferior nasal concha, and decrease in total IgE titer in blood were observed.

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US20100055082A1 (en) * 2008-09-04 2010-03-04 Jacques Alain Bauer Immunomodulatory extracts from lactobacillus bacteria and methods of manufacturing and use thereof
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US8226937B2 (en) 2006-12-21 2012-07-24 Calpis Co., Ltd. Agents for promoting IgA production
RU2460781C2 (ru) * 2009-01-29 2012-09-10 Общество с ограниченной ответственностью "ИнноПроб" (ООО "ИнноПроб") Иммунобиологическое противоаллергическое средство (варианты), штамм lactobacillus acidophilus, используемый при приготовлении иммунобиологического противоаллергического средства
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US10030274B2 (en) 2013-04-11 2018-07-24 Asahi Group Holdings, Ltd. Method for screening lactic acid bacteria having immunoregulatory function
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JP4688457B2 (ja) * 2004-09-10 2011-05-25 四国乳業株式会社 免疫増強組成物
JP4823503B2 (ja) * 2004-10-14 2011-11-24 ジェンモント バイオテック インコーポレイテッド Ｌａｃｔｏｂａｃｉｌｌｕｓｆｅｒｍｅｎｔｕｍの新規微生物株ＧＭ−０９０、およびＩＦＮ−γ分泌の刺激および／またはアレルギーの処置のためのその使用。
US7351572B2 (en) 2004-10-15 2008-04-01 Genmont Biotech Inc. Microorganism strain GM-090 of Lactobacillus fermentum and its use for stimulating IFN-γ secretion and/or treating allergy
TW200637908A (en) 2005-01-04 2006-11-01 Calpis Co Ltd Method for preparation of lactic acid bacterium having anti-allergic activity
EP1961309A4 (en) 2005-12-06 2010-01-20 Otsuka Pharma Co Ltd EMBRYONIC AXIS FERMENTATION PRODUCT OF SOYBEAN CONTAINING EQUOL, AND METHOD FOR PRODUCING THE SAME
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RU2431663C2 (ru) * 2008-01-28 2011-10-20 Общество с ограниченной ответственностью "ИнноПроб" (ООО "ИнноПроб") ИММУНОБИОЛОГИЧЕСКОЕ ПРОТИВОАЛЛЕРГИЧЕСКОЕ СРЕДСТВО (ВАРИАНТЫ) И ШТАММ Lactobacillus acidophilus 100 аш ПА, ИСПОЛЬЗУЕМЫЙ ДЛЯ ПРОИЗВОДСТВА ИММУНОБИОЛОГИЧЕСКОГО ПРОТИВОАЛЛЕРГИЧЕСКОГО СРЕДСТВА
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