US20030049231A1 - Agent for the anti-adhesion of skin pathogenic flora - Google Patents

Agent for the anti-adhesion of skin pathogenic flora Download PDF

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Publication number
US20030049231A1
US20030049231A1 US10/177,589 US17758902A US2003049231A1 US 20030049231 A1 US20030049231 A1 US 20030049231A1 US 17758902 A US17758902 A US 17758902A US 2003049231 A1 US2003049231 A1 US 2003049231A1
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Prior art keywords
composition
bacterial agent
bacterium
cfu
adhesion
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US10/177,589
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Inventor
Markus Baur
Ralf Zink
Isabelle Auzanneau
Karine Buffard
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Nestec SA
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Nestec SA
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Assigned to NESTEC S.A. reassignment NESTEC S.A. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BUFFARD, KARINE, AUZANNEAU, ISABELLE, ZINK, RALF, BAUR, MARKUS
Publication of US20030049231A1 publication Critical patent/US20030049231A1/en
Priority to US11/217,930 priority Critical patent/US8685389B2/en
Priority to US14/083,098 priority patent/US9226943B2/en
Priority to US14/957,889 priority patent/US20160082052A1/en
Abandoned legal-status Critical Current

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    • 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
    • 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
    • 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
    • A61K35/745—Bifidobacteria
    • 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
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A61K9/0017—Non-human animal skin, e.g. pour-on, spot-on
    • 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
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • A61P17/04—Antipruritics
    • 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
    • A61P17/08—Antiseborrheics
    • 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
    • A61P17/10—Anti-acne 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
    • 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
    • A61P31/10—Antimycotics
    • 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
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/005—Antimicrobial preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/007—Preparations for dry skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/10—Washing or bathing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • A61Q5/006—Antidandruff preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • A61Q5/02—Preparations for cleaning the hair
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/005—Preparations for sensitive skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/008—Preparations for oily skin

Definitions

  • the present invention relates to the use of a bacterial agent selected for its properties of anti-adhesion of skin pathogens, for the preparation of compositions which are for cosmetic, pharmaceutical or veterinary use and which are intended to stabilize and/or regulate the cutaneous ecosystem of mammals, and to the compositions containing such an agent.
  • pathogens such as Staphylococcus aureus, Streptococcus pyogenes or Propionibacterium acnes, or of certain yeasts
  • pathogens such as Staphylococcus aureus, Streptococcus pyogenes or Propionibacterium acnes, or of certain yeasts
  • pathogens such as Staphylococcus aureus, Streptococcus pyogenes or Propionibacterium acnes, or of certain yeasts
  • pathogens such as Staphylococcus aureus, Streptococcus pyogenes or Propionibacterium acnes, or of certain yeasts
  • antibiotics or chemical antibacterial agents are known. They are, for example, compositions based on aldehydes and derivatives.
  • French patent application FR 2740039 describes the use of a substance chosen from aldehydes and bifunctional compounds, preferably glutaraldehyde, for inhibiting the attachment of strains of pathogens such as Staphylococcus aureus to keratinocytes and corneocytes.
  • hexachlorophene and its derivatives are known as antibacterial substances and are more particularly used against Propionibacterium acnes.
  • PCT application WO 97/366603 demonstrates the antifungal properties of a strain of Lactobacillus casei.
  • Bacillus can also be used on skin or mucous membranes.
  • Bacillus coagulans Bacillus subtilis, Bacillus laterosporus and Bacillus laevolacticus are used in compositions intended to prevent bacterial, viral or fungal infections of skin or of mucous membranes.
  • the invention proposes to find a novel bacterial agent capable of controlling and regulating the cutaneous ecosystem in order to improve upon the deficiencies of the prior art.
  • the present invention relates to the use of a bacterial agent for preparing a composition which is for cosmetic, pharmaceutical or veterinary use and to the resulting compositions. These compositions are intended to be administered to humans or to animals for the purpose of preventing or treating disorders induced by pathogens of the cutaneous system.
  • the bacterial agent is generally an extract of a lactic acid bacterium, or a lactic acid bacterium, and is selected for its properties of adhesion to skin cells as well as for regulation of the attachment of skin pathogens, in particular by inhibiting their adhesion.
  • Suitable bacterial agents may be selected from strains of Lactobacillus, Micrococcus or Bifidobacterium, and preferably from the Lactobacillus johnsonii CNCM I-1225, Micrococcus varians CNCM I-1586, Micrococcus varians CNCM I- 1587 or Bifidobacterium lactis ATCC 27536 strains.
  • the bacterial strain can be used in a viable, deactivated or semi-active form. It also can be used in the form of a lyophilized powder, which can, e.g., comprise approximately 10 ⁇ 10 8 to 10 ⁇ 10 11 cfu/g.
  • compositions of the present invention are intended for cosmetic, pharmaceutical or veterinary use and contain at least one bacterial agent capable of stabilizing and/or of regulating the pathogenic flora of the cutaneous system.
  • the bacterial agent is an extract of a bacterium, or a bacterium, selected for its properties of adhesion to skin cells and its anti-adhesive properties with respect to pathogens of the cutaneous system.
  • compositions can also be used in ophthalmology or for nasal application. Also, they can in particular be in the form of a cream, lotion, hypoallergenic cleansing bar, shampoo or powder.
  • skin cells i.e. keratinocytes
  • corneocytes are grouped together under the name “cutaneous system”.
  • the present invention provides a bacterial agent selected for its property of adherence to skin cells, and of stabilization and regulation of the pathogenic bacterial flora of the cutaneous system, in particular by inhibiting the adhesion of pathogens such as Staphylococcus aureus, Streptococcus pyogenes or Propionibacterium acnes.
  • pathogens such as Staphylococcus aureus, Streptococcus pyogenes or Propionibacterium acnes.
  • pathogens such as Staphylococcus aureus, Streptococcus pyogenes or Propionibacterium acnes.
  • strains of Lactobacillus, of Micrococcus and of Bifidobacterium have been found to be useful, with a strain of Lactobacillus johnsonii (NCC 533), two strains of Micrococcus varians (NCC 1482, NCC 1520) and a strain of Bifidobacterium lactis (ATCC 27536) being preferred for selection as the agent.
  • the strain of Bifidobacterium lactis (ATCC 27536) can be obtained from Hansen (Chr. Hansen A/S, 10-12 Boege Alle, P.O. Box 407, DK-2970 Hoersholm, Denmark).
  • CNCM I-1586 NCC 1482
  • CNCM I-1587 NCC 1520
  • Gram-positive microorganism is permanently immobile.
  • Spherical form is in the form of irregularly arranged tetrades.
  • the bacteria according to the invention are used for preparing compositions intended for the prophylaxis or the treatment of disorders linked to pathogens of the cutaneous system, such as Staphylococcus aureus, Streptococcus pyogenes or Propionibacterium acnes, or yeasts.
  • disorders linked to pathogens of the cutaneous system such as Staphylococcus aureus, Streptococcus pyogenes or Propionibacterium acnes, or yeasts.
  • These skin disorders can be in particular atopic dermatitis (in the remission phases, as a maintenance treatment), acne, candidiases, seborrhoeic dermatitis, pityriasis versicolor, impetigo or eczematous secondary infections.
  • the disorders of the cutaneous system may also be linked to therapies with antibiotics or antimycotic agents, to diabetes (candidiases), to a pathology of mucous membranes (vaginal candidiasis), to chronic eczema (homeostasis imbalance), to sensitive skin (premature babies, children) or greasy skin (linked to hormonal dysregulation which may promote the growth of bacteria) or to dandruff.
  • therapies with antibiotics or antimycotic agents to diabetes (candidiases), to a pathology of mucous membranes (vaginal candidiasis), to chronic eczema (homeostasis imbalance), to sensitive skin (premature babies, children) or greasy skin (linked to hormonal dysregulation which may promote the growth of bacteria) or to dandruff.
  • the bacteria according to the invention can be used in their live or semi-active form, or in a deactivated form.
  • the expression “bacterium in a semi-active form” is intended to mean a bacterium with low physiological activity. This activity can be measured by a longer exponential growth phase or generation time, a metabolism which has slowed or an incomplete physiological response to modifications of the environment, for example. In certain extreme cases, the number of bacteria may be decreased since they can no longer withstand the change in the environment.
  • Bacterial culture supernatants can also be used successfully in this invention.
  • the bacterial agent can be an extract of a bacterium, or a bacterium itself in a viable active form.
  • the bacterial agent is then preferably converted into a lyophilized powder, for example, according to the method described in EP 818529.
  • the powder typically contains from 10 ⁇ 10 8 to 10 ⁇ 10 11 cfu/g.
  • the bacterial agent can be an extract of a bacterium, or the bacterium itself in a semi-active form.
  • the partial deactivation of the strains can be carried out in several ways, in particular by:
  • freeze drying consisting of cycles of freezing in liquid nitrogen/thawing at 37° C. A decrease of approximately 1 log can then be obtained
  • the bacterial agent can then be used in the form of a powder containing at least 10 ⁇ 10 6 cfu/g, and preferably in dry compositions, such as dry shampoos or other powder compositions, which can contain up to 10% of the bacterial extract.
  • the bacterial agent can also be an extract of a bacterium, or a bacterium, in a deactivated form.
  • the bacterium is preferably inactivated by heat treatment at approximately 90° C. for approximately 2 hours.
  • the bacterial agent is in the form of a lyophilized powder containing from 10 ⁇ 10 8 to 10 ⁇ 10 12 cfu/g. It can be used at up to 5%, and from preferably from 0.05 to 3%, in liquid compositions and at up to 10% in pulverulent compositions.
  • the present invention also relates to a composition which is for cosmetic, pharmaceutical or veterinary use and which contains a bacterial agent having the properties as described above.
  • At least one bacterial strain in viable, semi-active or deactivated form is incorporated into a pharmaceutically or cosmetically acceptable support in an amount which varies as a function of the desired use.
  • the bacterial agent can be present at up to approximately 5% with respect to the total weight of the composition and at up to 10% for compositions in the form of a powder, and preferably at between 0.5 to 2%.
  • compositions according to the invention can be administered via the topical or ocular route.
  • the pharmaceutical compositions based on compounds according to the invention are preferably intended for the treatment of skin and of mucous membranes, and can be in the form of salves, of creams, of milks, of ointments, of powders, of soaked swabs, of solutions, of gels, of sprays, of lotions or of suspensions. They can also be in the form of microspheres or nanospheres, or lipid or polymeric vesicles, or of polymer patches and of hydrogels, which allow controlled release.
  • These compositions for administration via the topical route can be either in anhydrous form or aqueous form, depending on the clinical indication.
  • the invention relates to a cosmetic composition containing, in a cosmetically acceptable support, at least one bacterial agent as defined above.
  • the cosmetic composition can contain the bacterial agent in a proportion of at least 0.001% by weight with respect to the total weight of the composition, and preferably from 0.05 to 3%.
  • This cosmetic composition is in particular intended for body and hair hygiene. It can in particular be in the form of a cream, a milk, a lotion, a gel, microspheres or nanospheres, or lipid or polymeric vesicles, a soap or a shampoo.
  • the viable or inactivated bacterial agent can be combined with retinoids or corticosteroids, or combined with anti-free radicals, with ⁇ -hydroxy or ⁇ -keto acids or their derivatives, or with ion channel blockers.
  • the pharmaceutical and cosmetic compositions according to the invention can also contain inert additives or even pharmacodynamically or cosmetically active additives, or combinations of these additives, and in particular: wetting agents; depigmenting agents such as hydroquinone, azelaic acid, caffeic acid or kojic acid; emollients; moisturizing agents such as glycerol, PEG-400, thiamorpholinone and its derivatives, or urea; anti-seborrhoeic or anti-acne agents, such as S-carboxymethylcysteine, S-benzylcysteamine, their salts and their derivatives, or benzoyl peroxide; antibiotics such as erythromycin and its esters, neomycin, clindamycin and its esters; tetracyclins; antifungal agents such as ketoconazole or 4,5-polymethylene-3-isothiazolinones; agents for promoting the regrowth of
  • composition according to the invention can also contain preserving agents such as para-hydroxybenzoic acid esters, stabilizers, moisture regulators, pH regulators, osmotic pressure modifiers, emulsifying agents, UV-A and UV-B screening agents, and antioxidants such as ⁇ -tocopherol, butylhydroxyanisole or butylhydroxytoluene.
  • preserving agents such as para-hydroxybenzoic acid esters, stabilizers, moisture regulators, pH regulators, osmotic pressure modifiers, emulsifying agents, UV-A and UV-B screening agents, and antioxidants such as ⁇ -tocopherol, butylhydroxyanisole or butylhydroxytoluene.
  • the present invention also relates to a composition which is for veterinary or cosmetic use for animals and which contains at least one bacterial agent as defined above.
  • a composition which is for veterinary or cosmetic use for animals and which contains at least one bacterial agent as defined above.
  • Such a composition can be in the form of dry or liquid shampoos, powders, foams or lotions, for example. It can contain up to 10% of the bacterial agent.
  • composition according to the invention is intended in particular for the therapeutic or prophylactic treatment of healthy, sensitive and/or diseased skin and/or mucous membranes which may exhibit disorders of the cutaneous system, such as in particular:
  • infectious complications such as superinfected atopic dermatitis, impetigo-based eczema, ulcers, wounds, bums, superinfected inflammatory acne,
  • dermatitises such as impetigo, superficial folliculitis,
  • dermatophytoses (Tinea capitis, Tinea corporis, athlete's foot, Hebra's eczema, herpes carcinatus),
  • candidiases vaginal, interdigital, linked to professions at risk or to diabetes
  • compositions for veterinary use are particularly intended to treat or prevent dysfunctions due to staphylococcal infections (due to Staphylococcus aureus, S. intermedians ), streptococcal infections (due to S. pyogenes ) and mycotic infections (candidoses due to C. albicans and pytirosporoses due to P. canis ).
  • the bacterial cultures (1 ml) are incubated in 10 ml of medium (cf. Table 1) overnight.
  • the bacteria are precultured until a concentration of 5.0 ⁇ 10 8 to 10 9 cfu/ml is obtained.
  • the cfu are standardized by measuring the optical density of each strain (OD at 10 8 cfu/ml: see Table 1).
  • the bacterial strains are assayed for their adhesion properties.
  • SV40 T-Ag immortalized cell lines DK2-NR and FK2-NR cells as described in EP 780 469 and,
  • HPV Human papilloma virus
  • E6/E7 and SV40 T-Ag immortalized cell lines DK7-NR cell lines as described in application WO 99/02347.
  • the culture medium for the cell lines was as follows: DK7-NR, FK2-NR: NR-2 (Biofluids, Rockville, Md. 20850) (EP 780469). DK2-NR: NR-M (Biofluids, Rockville, Md. 20850).
  • the keratinocyte lines are cultured in a proportion of 5 ⁇ 10 5 keratinocytes/cm 2 , seeded in coated 6-well clusters (Becton Dickinson, Lincoln Park, N.J.).
  • the coating solution consists of basic medium supplemented with 10 ⁇ g/ml of human fibronectin (Becton Dickinson), 31 ⁇ g/ml of bovine collagen I (Vitrogen, Collagen Corporation, Fremont, Calif.) and 0.1 mg/ml of BSA (Biofluids, Rockville, Md. 20850).
  • the cell cultures form a monolayer (confluent).
  • the Ca 2+ concentration of the medium is brought to 1.5 mM so as to induce cell differentiation.
  • the cells are cultured for 4 days in a high calcium concentration medium, without antibiotics.
  • the adhesion assays the cell cultures are washed 3 times with buffer (HBSS, Ca 2+ : 1.0 mM).
  • the bacterial strains are labelled overnight by adding 100 ⁇ Ci/10 ml of 2- 3 H-adenine (Amersham, TRK.311) to the culture medium. Aliquot fractions of the bacteria are incubated in a medium without 3 H-adenine. The unlabelled (cold) supernatant is set aside in order to adjust the cfu/ml for the adhesion assays.
  • the bacterial suspensions are centrifuged for 10 minutes are 4000 rpm. Before adjusting the optical density (OD), the pellets are washed twice in HBSS. The OD is measured for each strain so as to adjust the final concentrations of bacteria to 10 6 , 10 7 , 10 8 and 10 9 cfu/ml.
  • the medium for the adhesion assays is a 1:1 mixture of keratinocyte culture medium and of the unlabelled supernatant of the bacterial medium.
  • the bacterial suspensions are incubated on plastic dishes and plastic dishes coated with cells.
  • the adhesion index (AI) is calculated as 3 H activity (dpm/well), as % of the total 3 H activity (dpm/ml) of the bacterial suspension.
  • the adhesion index of the 13 different bacterial strains is calculated by measuring the 3 H adenine activity of the radiolabelled microorganisms. The results are given in Table 2.
  • Immortalized human keratinocytes of the HaCaT line are used (Boukamp P. et al., J. Cell Biol., 106, 761-771, 1988).
  • the HaCaT cells are cultured in DMEM medium supplemented with 10% of foetal calf serum, at 37° C. under 5% of CO 2 .
  • 6-well clusters (Becton Dickinson) are seeded in a proportion of 10 4 cells/cm 2 . After 4 to 5 days, the cells reach confluence. The adhesion assays are carried out 4 to 5 days after confluence. The monolayers are washed 3 times with PBS before these assays.
  • the bacteria are labelled with 2- 3 H-adenine (Amersham, TRK 311), in a proportion of 100 ⁇ Ci/10 ml of broth.
  • the suspensions are washed 3 times and then resuspended in PBS.
  • the cell density is adjusted in this same buffer.
  • the adhesion is defined by the ratio between radioactivity which has adhered and radioactivity which was introduced, multiplied by 100.
  • the radiolabelled pathogen and the cold bacterial strain are incubated simultaneously with the monolayer.
  • the assays are carried out in triplicate for bacterial agent densities covering 3 logs.
  • the in vitro adhesion model is based on the incubation of a radiolabelled and calibrated suspension of a skin pathogenic microorganism ( Staphylococcus aureus ) with a monolayer of immortalized human keratinocytes (HaCaT line) (Boukamp P. et al., J. Cell Biol., 106, 761-771, 1988).
  • a skin pathogenic microorganism Staphylococcus aureus
  • HaCaT line monolayer of immortalized human keratinocytes
  • the inhibitory activity of the bacterial agent ( Lactobacillus johnsonii NCC 533 in a viable or deactivated form) with respect to this adhesion is evaluated in the context of a co-incubation, on the monolayer, of the pathogen and of the compound to be assayed, by measuring the radioactivity retained on the monolayer.
  • the HaCaT cells are cultured in DMEM supplemented with 10% of foetal calf serum, at 37° C. under 5% of CO 2 . They are seeded in 6-well clusters in a proportion of 10 4 cells/cm 2 .
  • the adhesion assay is carried out 5 days after confluence. The monolayers are washed 3 times with PBS before incubation with the microorganisms.
  • Staphylococcus aureus (ATCC 6538) is cultured in TCS medium, in aerobiosis at 35° C.
  • Lactobacillus johnsonii NCC 533 is cultured in MRS medium, in anaerobiosis at 37° C.
  • the deactivated form of NCC 533 is obtained by lyophilizing a dense suspension of Lactobacilli which has been subjected to several cycles of freezing in liquid nitrogen/thawing at room temperature.
  • the preparation assayed corresponds to a biomass of 4.0 ⁇ 10 10 cfu/g.
  • mice 15 7- to 8-week-old SKH female mice weighing approximately 30 g were supplied by C. River. 5 mice were used for each group assaying a different topical application.
  • Microorganisms A strain of Staphylococcus aureus (named: strain 1) which was isolated from a human skin lesion (leg ulcer) is used. This strain is sensitive to methycilin.
  • a suspension of the bacterium is prepared for inoculation in the mice.
  • a preculture in the exponential growth phase of strain 1 is prepared on a solid medium (AES, AEB 122 859) at 35° C. for 18 to 24 h.
  • the bacterium is resuspended in 10 ml of sterile saline solution, and then recovered after centrifugation at 3000 [lacuna] for 10 min. The supernatant is then removed and the pellet is taken up with 10 ml of saline solution. This procedure is repeated twice.
  • An inoculum suspension is prepared by resuspending the washed bacteria in 4 ml of sterile saline solution. The OD at 525 nm is adjusted to approximately 0.14. It contains approximately 10 8 cfu/ml.
  • mice The skin of the mice is delipidized on the flanks with 95 [sic] ethanol (Merck). 50 ⁇ l of a suspension containing a 50/50 mixture of the S. aureus inoculum, 10 7 cfu/ml, and of the product to be assayed were slowly applied to the delipidized area (6.25 cm 2 ), using a micropipette. The inoculated sites are protected by occlusion for 1 h under a sterile plastic dressing (Dermafilm 33 ⁇ 15, ref. 38.3015, Vygon laboratory).
  • mice 4 hours after application of the suspension, the mice are killed under anaesthesia with forene (Abbott France).
  • the inoculated sites are excised as a block (12 mm diameter).
  • the skin biopsies removed are ground and homogenized with 2 ml of sterile saline solution, using a Polytron (PT 2100, Bioblock Scientific) (5 rpm, 5 min.).
  • a 1 ml sample of the homogenized tissue is added to 9 ml of a sterile saline solution, and 0.1 ml of this mixture is cultured on a staphylococcal medium No. 110 using the 10-fold dilutron method. After 48 hours of incubation at 35° C., the colonies developed are counted and the CFU(colony forming units) are determined.
  • NCC 533 at 0.5% and 1% decreases the number of S. aureus bacteria found by approximately 1 log for the inocula at 10 6 cfu/ml and 10 7 cfu/ml. No dose effect is observed, either with the 10 6 cfu/ml inoculum or with the inoculum at 10 7 cfu/ml.
  • a body lotion is prepared which has the following composition: 8.0% of mineral oil, 5.0% of isopropyl palmitate, 2.0% of polyglyceryl-3 diisostearate, 4.0% of octyldodecanol, 0.3% of carbomer, 0.2% of sodium cocoylglutamate, 1.2% of 10% sodium hydroxide, a preserving agent, fragrance, 0.5 to 3% of a lyophylisate containing from 10 ⁇ 10 8 to 10 ⁇ 10 12 cfu/g of at least one bacterial strain chosen from Lactobacillus johnsonii (CNCM I-1225), Micrococcus varians (CNCM I-1586 or CNCM I-1587) or Bifidobacterium lactis (ATCC 27536, Hansen) and inactivated by heat treatment at approximately 90° C. for about 2 hours. The mixture is made up to 100% with water.
  • the body lotion thus obtained is intended, due to its anti-adhesion properties with respect to pathogens, to stabilize and/or regulate skin pathogenic flora.
  • a shampoo is prepared which has the following composition: 7.0% of sodium lauryl sulphate, 2.0% of cocamidopropyl betaine, 2.0% of sodium lauryl sulphonosuccinate, sodium chloride, preserving agent, fragrance and from 0.5 to 3% of a lyophilisate containing from 10 8 to 10 12 cfu/g of at least one bacterial strain chosen from Lactobacillus johnsonii (CNCM I-1225), Micrococcus varians (CNCM I-1586 or CNCM I-1587) or Bifidobacterium lactis ATCC 27536, and inactivated by heat treatment at approximately 90° C. for about 2 hours. The mixture is made up to 100% with water.
  • the shampoo thus prepared has properties which regulate scalp pathogenic flora. It is in particular indicated in the treatment of dandruff.
  • fatty and aqueous phases are prepared which have the following composition: L. johnsonii (CNCM I-1225) 1% as described in Example 5
  • Fatty phase Arachidyl behenyl alcohol/ 3% arachidylglucoside Isohexadecane 7% Sweet almond oil 3% Karite butter 2% B.H.T. 0.05%
  • Aqueous phase Water Qs 100% Glycerol 5% Methyl POB 0.1%
  • the fatty and aqueous phases are heated to 75° C. Then, emulsification is carried out by adding the aqueous phase to the fatty phase with Rayneri mixing at 1000 rpm. 30 minutes after the emulsification, the mixture is homogenized for 1 minute with a polytron (speed 4-5).
  • Example 7 In the same way as in Example 7, a composition is prepared which has the following composition: L. johnsonii (CNCM I-1225) 1% as described in Example 5 Fatty phase: Glyceryl stearate and 5% PEG100 stearate Isohexadecane 8% Karite butter 5% B.H.T. 0.05% DC 1503 1% Aqueous phase: Water Qs 100% Glycerol 3% Carbopol 981 0.2% Lubrajel 5% Phenoxyethanol 1% Sodium hydroxide Qs pH 6
  • Example 7 In the same way as in Example 7, a composition is prepared which has the following composition: L. johnsonii (CNCM I-1225) 1% as described in Example 5 Fatty phase: Polyglyceryl-3 diisostearate 5% Cyclomethicone CM5 20% Aqueous phase: Water Qs 100% Glycerol 5% NaCl 0.5% MgSO4 0.5%
  • a shampoo for pets is prepared which has the following composition: 5% of sodium lauryl sulphate, 2% of cocamidopropyl betaine, 2% of sodium lauryl sulphonosuccinate, 2% of sodium chloride, 1.5% of PEG-7 glyceryl cocoate, 0.75% of propylene glycol, panthenol, glycerol, disodium phosphate, preserving agent, fragrance and 1% of L. johnsonii (CNCM I-1225) as described in Example 5. The mixture is made up to 100% with water.
  • the shampoo thus prepared has properties which regulate the pathogenic flora of the cutaneous system of pets.

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US9226943B2 (en) 2016-01-05
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ATE471152T1 (de) 2010-07-15
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US20160082052A1 (en) 2016-03-24
RU2002119396A (ru) 2004-02-27
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CN1434716B (zh) 2012-03-28
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