U.S. patent application number 10/177589 was filed with the patent office on 2003-03-13 for agent for the anti-adhesion of skin pathogenic flora.
Invention is credited to Auzanneau, Isabelle, Baur, Markus, Buffard, Karine, Zink, Ralf.
Application Number | 20030049231 10/177589 |
Document ID | / |
Family ID | 8241061 |
Filed Date | 2003-03-13 |
United States Patent
Application |
20030049231 |
Kind Code |
A1 |
Baur, Markus ; et
al. |
March 13, 2003 |
Agent for the anti-adhesion of skin pathogenic flora
Abstract
Bacterial agents for preparing compositions which are for
cosmetic, pharmaceutical or veterinary use and which are intended
to stabilize and/or regulate the cutaneous ecosystem of mammals.
These bacterial agents are extracts of a bacterium, or a bacterium
and are selected for their adhesion to skin cells and anti-adhesion
to pathogens of the cutaneous system. The invention also relates to
compositions containing such agents.
Inventors: |
Baur, Markus; (Stuttgart,
DE) ; Zink, Ralf; (Le Mont Pelerin, CH) ;
Auzanneau, Isabelle; (Opio, FR) ; Buffard,
Karine; (Sevres, FR) |
Correspondence
Address: |
WINSTON & STRAWN
PATENT DEPARTMENT
1400 L STREET, N.W.
WASHINGTON
DC
20005-3502
US
|
Family ID: |
8241061 |
Appl. No.: |
10/177589 |
Filed: |
June 21, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10177589 |
Jun 21, 2002 |
|
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PCT/EP00/12719 |
Dec 13, 2000 |
|
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Current U.S.
Class: |
424/93.4 ;
424/780 |
Current CPC
Class: |
A61Q 19/00 20130101;
A61P 31/00 20180101; A61Q 19/005 20130101; A61K 35/74 20130101;
A61Q 19/007 20130101; A61P 31/10 20180101; A61P 17/10 20180101;
A61P 17/00 20180101; A61Q 19/008 20130101; A61K 35/745 20130101;
A61K 9/0014 20130101; A61Q 5/006 20130101; A61K 35/744 20130101;
A61P 17/02 20180101; A61Q 5/02 20130101; A61P 17/08 20180101; A61Q
17/005 20130101; A61P 31/04 20180101; A61K 9/0017 20130101; A61K
35/747 20130101; A61P 17/04 20180101; A61P 37/08 20180101; A61Q
19/10 20130101; A61K 8/99 20130101 |
Class at
Publication: |
424/93.4 ;
424/780 |
International
Class: |
A61K 035/66 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 22, 1999 |
EP |
99204489.1 |
Claims
What is claimed is:
1. A method of preparing compositions for cosmetic, pharmaceutical
or veterinary use and which are intended to be administered to
humans or to animals for the therapeutic or prophylactic purpose of
stabilizing and/or regulating the pathogenic flora of the cutaneous
system, which comprises associating a bacterial agent with the
composition, the agent being 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.
2. The method of claim 1 which further comprises providing the
agent in the composition in an amount of at least 10.times.10.sup.6
cfu/g.
3. The method of claim 1, wherein the bacterial agent is selected
from the species of Lactobacillus, Micrococcus or
Bifidobacterium.
4. The method of claim 1, wherein the bacterial agent is
Lactobacillus johnsonii CNCM I-1225, Micrococcus varians CNCM
I-1586, Micrococcus varians CNCM I-1587 or Bifidobacterium lactis
ATCC 27536.
5. The method of claim 1, wherein the bacterium is in a viable,
semi-active or deactivated form.
6. The method of claim 5, wherein the bacterium is deactivated by a
heat treatment at 90.degree. C. for 2 hours.
7. The method of claim 5, wherein the bacterial agent is present in
a lyophilized form that contains from 10.sup.8 to 10.sup.11
cfu/g.
8. A composition for cosmetic, pharmaceutical or veterinary use and
which contains at least one bacterial agent capable of stabilizing
and/or of regulating the pathogenic flora of the cutaneous system,
with the bacterial agent being an extract of a bacterium, or a
bacterium, and is selected for its properties of adhesion to skin
cells and its anti-adhesive properties to pathogens of the
cutaneous system.
9. The composition of claim 8, in which the bacterial agent is a
species of Lactobacillus, Micrococcus or Bifidobacterium.
10. The composition of claim 8, wherein the bacterial agent is
Lactobacillus johnsonii CNCM I-1225, Micrococcus varians CNCM
I-1586, Micrococcus varians CNCM I-1587 or Bifidobacterium lactis
ATCC 27536.
11. The composition of claim 8, wherein the agent is present in the
composition in an amount of at least 10.times.10.sup.6 cfu/g.
12. The composition of claim 8, wherein the bacterium is in a
viable, semi-active or deactivated form.
13. The composition of claim 12, wherein the bacterial agent is
present in a lyophilized form that contains from 10.sup.8 to
10.sup.11cfu/g.
14. The composition of claim 12, in which the bacterium is
deactivated by heat treatment at 90.degree. C. for 2 hours.
15. The composition of claim 12, which contains up to 10% by weight
of the bacterial agent with respect to the total weight of the
composition.
16. The composition of claim 12, which contains approximately 0.05
to 5% by weight of the bacterial agent with respect to the total
weight of the composition.
17. A method for the treatment or prophylaxis of skin disorders
which comprises administering to a subject in need of such
treatment or prophylaxis a composition according to claim 8.
18. The method of claim 17 wherein the skin disorder is one or more
of: infectious complications such as superinfected atopic
dermatitis, impetigo-based eczema, ulcers, wounds, bums,
superinfected inflammatory acne, dermatitises such as impetigo,
superficial folliculitis, seborrhoeic dermatitises, pityriasis
versicolor, dermatophytoses, candidiases, those linked to therapies
with antibiotics or to antimycotic agents, those caused by hormonal
dysregulation, or dandruff.
19. The method of claim 17, wherein the composition is applied to
sensitive skin or greasy skin.
20. A veterinary composition comprising a composition according to
claim 8, intended for the treatment or for the prevention of
dysfunctions linked to staphylococcal, streptococcal and mycotic
infections in animals.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of the US national stage designation
of International Application PCT/EP00/12719 filed Dec. 13, 2000,
the entire content of which is expressly incorporated herein by
reference thereto.
TECHNICAL FIELD
[0002] 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.
BACKGROUND ART
[0003] The proliferation of pathogens such as Staphylococcus
aureus, Streptococcus pyogenes or Propionibacterium acnes, or of
certain yeasts, can lead to dysregulation of the cutaneous system,
or even more serious disorders of skin or of mucous membranes, such
as eczema, candidiases, dermatitises, etc.
[0004] Many means of treatment against these pathogenic agents are
known. The most conventionally used are antibiotics or chemical
antibacterial agents. They are, for example, compositions based on
aldehydes and derivatives.
[0005] Thus, the 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.
[0006] Thus, hexachlorophene and its derivatives are known as
antibacterial substances and are more particularly used against
Propionibacterium acnes.
[0007] However, these treatments are in general expensive and
harmful to both the health and the environment. Alternatively,
nontoxic treatments are now known which consist in using the
antifungal, bactericidal or bacteriostatic properties of certain
strains of microorganisms.
[0008] Thus, PCT application WO 97/366603 demonstrates the
antifungal properties of a strain of Lactobacillus casei.
[0009] Other bacterial agents, such as the Bacillus, can also be
used on skin or mucous membranes. Specifically, in application WO
98/47374, strains of 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.
[0010] 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.
SUMMARY OF THE INVENTION
[0011] 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.
[0012] 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.
[0013] 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.times.10.sup.8
to 10.times.10.sup.11 cfu/g.
[0014] The 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. As noted above, 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.
[0015] These 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.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0016] In this invention, skin cells (i.e. keratinocytes) as well
as corneocytes are grouped together under the name "cutaneous
system"
[0017] 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. For this, many bacterial strains were
tested for their properties of attachment to human keratinocytes
(cf., Example 1).
[0018] From diverse bacterial strains thus tested, 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.
[0019] The strain of Lactobacillus johnsonii (NCC 533) and the
strains of Micrococcus varians (NCC 1482 and NCC 1520) were
deposited, according to the Treaty of Budapest, at the Collection
Nationale de Cultures de Microorganismes (CNCM) [National
Collection of Microorganism Cultures], Institut Pasteur, 28 rue du
Docteur Roux, 75724 Paris Cedex 15, France, respectively on Jun.
30, 1992 under the reference CNCM I-1225 for Lactobacillus
johnsonii, and Jun. 07, 1995 under the references and CNCM I-1586
and CNCM I-1587 for Micrococcus varians NCC 1482 and NCC 1520.
[0020] 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).
[0021] Details concerning the morphology and the general properties
of the strains are given below:
Lactobacillus johnsonii CNCM I-1225
[0022] Morphology
[0023] Non-motile Gram-positive microorganism which does not form
spores.
[0024] Fairly short and squat isolated rods.
[0025] Metabolism
[0026] Microaerophilic microorganism with homofermentative
metabolism giving rise to the production of L (+) and D (-) lactic
acid.
[0027] Other characteristics: Catalase (-), CO.sub.2 production
(-), arginine hydrolysis (-).
[0028] Fermentation of Sugars:
[0029] Amygdalin (+), arabinose (-), cellobiose (+), esculin (+),
fructose (+), galactose (-), glucose (+), lactose (+), maltose
(+/-), mannitol (-), mannose (+), melibiose (-), raffinose (+),
ribose (-), salicine (+), sucrose (+), trehalose (+).
Micrococcus varians CNCM I-1586 (NCC 1482) and CNCM I-1587 (NCC
1520)
[0030] Morphology
[0031] Gram-positive microorganism, is permanently immobile.
[0032] Spherical form, is in the form of irregularly arranged
tetrades.
[0033] Metabolism
[0034] Aerobic microorganism, catalase (+)
[0035] Other characteristics: yellow colour on BHI medium. The
optimum growth temperature of said strains is 25-37.degree. C.
[0036] Fermentation of Sugars
[0037] Fructose (+), glucose (+).
[0038] 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. 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.
[0039] 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.
[0040] 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.
[0041] Bacterial culture supernatants can also be used successfully
in this invention.
[0042] According to a first embodiment, 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.times.10.sup.8
to 10.times.10.sup.11 cfu/g.
[0043] According to another embodiment, 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:
[0044] freeze drying, consisting of cycles of freezing in liquid
nitrogen/thawing at 37.degree. C. A decrease of approximately 1 log
can then be obtained,
[0045] the action of UV rays (15 to 60 minutes at 254 nm, distance
20 cm): decrease of 2 to 3 logs,
[0046] the action of heat (70.degree. C. for 3 hours): decrease of
approximately 3 to 4 logs, for example.
[0047] The bacterial agent can then be used in the form of a powder
containing at least 10.times.10.sup.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.
[0048] Finally, 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.degree. C. for approximately 2 hours. The bacterial agent is in
the form of a lyophilized powder containing from 10.times.10.sup.8
to 10.times.10.sup.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.
[0049] 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.
[0050] In order to prepare such compositions, 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%.
[0051] The compositions according to the invention can be
administered via the topical or ocular route.
[0052] Via the topical 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.
[0053] Via the ocular route, they are mainly used as eyewashes.
[0054] In a preferred embodiment, 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%.
[0055] 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.
[0056] In the compositions according to the invention, the viable
or inactivated bacterial agent can be combined with retinoids or
corticosteroids, or combined with anti-free radicals, with
.alpha.-hydroxy or .alpha.-keto acids or their derivatives, or with
ion channel blockers.
[0057] 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 hair, such as Minoxidil (2,4-diamino-6-piperidi-
nopyrimidine 3-oxide) and its derivatives, Diazoxide
(7-chloro-3-methyl-1,2,4-benzothiadiazine 1,1-dioxide) and
Phenytoin (5,4-diphenylimidazolidine-2,4-dione) [sic]; nonsteroidal
anti-inflammatory agents; carotenoids and, in particular,
.beta.-carotene; anti-psoriatic agents such as anthraline and its
derivatives; and finally, eicosa-5,8,11,14-tetraynoic acid and
eicosa-5,8,11-trynoic acid, their esters and amides.
[0058] The 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 .alpha.-tocopherol, butylhydroxyanisole or
butylhydroxytoluene.
[0059] Finally, 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. 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.
[0060] The 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:
[0061] infectious complications such as superinfected atopic
dermatitis, impetigo-based eczema, ulcers, wounds, bums,
superinfected inflammatory acne,
[0062] dermatitises such as impetigo, superficial folliculitis,
[0063] seborrhoeic dermatitises, pityriasis versicolor,
[0064] dermatophytoses (Tinea capitis, Tinea corporis, athlete's
foot, Hebra's eczema, herpes carcinatus),
[0065] candidiases (vaginal, interdigital, linked to professions at
risk or to diabetes),
[0066] disorders linked to therapies with antibiotics or to
antimycotic agents,
[0067] disorders caused by hormonal dysregulation (greasy skin) or
to [sic] dandruff,
[0068] sensitive skin (premature babies, children).
[0069] The 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).
EXAMPLE
[0070] Other characteristics of the present invention will appear
in the course of the following descriptions of examples of
embodiments, which are provided for the purposes of illustrating
the present invention but not to limit it.
Example 1
Selection of the Bacterial Agent
[0071] In the context of the present invention, the adhesion of 12
different bacterial strains to skin cells, in particular to HaCat
human keratinocytes in culture, is studied. These strains belong to
the Lactobacillus, Bifidobacterium, Micrococcus, Staphylococcus,
Streptococcus and Propionibacterium genera.
[0072] The bacterial cultures (1 ml) are incubated in 10 ml of
medium (cf. Table 1) overnight. For the adhesion assays, the
bacteria are precultured until a concentration of
5.0.times.10.sup.8 to 10.sup.9 cfu/ml is obtained. The cfu are
standardized by measuring the optical density of each strain (OD at
10.sup.8 cfu/ml: see Table 1).
[0073] Then, the bacterial strains are assayed for their adhesion
properties.
1TABLE 1 Bacterial strains and culture conditions OD at NCC
Incubation 10.sup.2 Bacterial strains code Medium T .degree.
C./hour cfu Lactobacillus johnsonii 533 MRS Anaerob. 37.degree. C./
1.00 La1 48 h Lactobacillus acidophilus 90 MRS Anaerob. 37.degree.
C./ 1.00 La10 48 h Bifidobacterium lactis 536 MRS Anaerob.
37.degree. C./ 0.65 ATCC27536 18 h Bifidobacterium longum 585 MRS
Aerob. 30.degree. C./18 h 1.32 B28 Micrococcus varians 1482 BHi
Aerob. 30.degree. C./18 h 4.85 NCC 1482 Micrococcus varians 1520
BHi Aerob. 30.degree. C./18 h 3.47 NCC 1520 Micrococcus varians
1583 BHi Aerob. 30.degree. C./18 h 4.18 MCV 17 Staphylococcus
carnosus 931 BHi Aerob. 30.degree. C./18 h 40.20 STC 21
Staphylococcus 751 BHi Aerob. 30.degree. C./18 h 3.26
piscifermentans STF4 Streptococcus 2019 HJL Aerob. 40.degree. C./18
h 0.37 thermophilus Sfi 16 Propionibacterium 1197 MRS Aerob.
30.degree. C./24 h 0.18 shermanii PP12 Propionibacterium 1116 MRS
Aerob. 30.degree. C./24 h 0.25 thoenii PP22 NCC 533, NCC 90, NCC
536 and NCC 585 were cultured under anaerobic conditions (Gaspack
H.sub.2 + CO.sub.2).
Human Keratinocyte Lines
[0074] The adhesion properties of the bacteria were studied on 3
keratinocyte lines:
[0075] SV40 T-Ag immortalized cell lines: DK2-NR and FK2-NR cells
as described in EP 780 469 and,
[0076] HPV (Human papilloma virus) E6/E7 and SV40 T-Ag immortalized
cell lines: DK7-NR cell lines as described in application WO
99/02347.
[0077] 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).
[0078] The keratinocyte lines are cultured in a proportion of
5.times.10.sup.5 keratinocytes/cm.sup.2, seeded in coated 6-well
clusters (Becton Dickinson, Lincoln Park, N.J.). The coating
solution consists of basic medium supplemented with 10 .mu.g/ml of
human fibronectin (Becton Dickinson), 31 .mu.g/ml of bovine
collagen I (Vitrogen, Collagen Corporation, Fremont, Calif.) and
0.1 mg/ml of BSA (Biofluids, Rockville, Md. 20850). After 6 to 8
days, the cell cultures form a monolayer (confluent). Next, the
Ca.sup.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. For the
adhesion assays, the cell cultures are washed 3 times with buffer
(HBSS, Ca.sup.2+: 1.0 mM).
Radiolabelling
[0079] The bacterial strains are labelled overnight by adding 100
.mu.Ci/10 ml of 2-.sup.3H-adenine (Amersham, TRK.311) to the
culture medium. Aliquot fractions of the bacteria are incubated in
a medium without .sup.3H-adenine. The unlabelled (cold) supernatant
is set aside in order to adjust the cfu/ml for the adhesion
assays.
Adhesion Assays
[0080] 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.sup.6,
10.sup.7, 10.sup.8 and 10.sup.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.
[0081] In order to analyse the adhesion properties of the bacteria
on a substrate without keratinocytes, the bacterial suspensions are
incubated on plastic dishes and plastic dishes coated with
cells.
Analysis
[0082] After washing the cultures 3 times with HBSS (Ca.sup.2+, 1.0
mM), the bacteria associated with the keratinocytes are lysed in a
solution of 1N NaOH for 30 minutes at room temperature. The
solution is transferred into scintillation vials with 1 ml of
benzethonium hydroxide (Sigma, St. Louis, USA). After 1 h at
60.degree. C., the .sup.3H activity of the label bacteria is
measured by liquid scintillation counting (dpm).
[0083] The adhesion index (AI) is calculated as .sup.3H activity
(dpm/well), as % of the total .sup.3H activity (dpm/ml) of the
bacterial suspension.
Results
[0084] (a) Adhesion index (AI)
[0085] The adhesion index of the 13 different bacterial strains is
calculated by measuring the .sup.3H adenine activity of the
radiolabelled microorganisms. The results are given in Table 2.
2TABLE 2 Adhesion index of bacterial strains of FK2-NR
keratinocytes dpm Adhesion index NCC Code CFU/ml (.times. 10.sup.3)
=SD (% of total dpm) 533 10.sup.9 209.6 24.2 1.2 10.sup.8 175.5
42.5 10.4 10.sup.7 39.5 1.9 23.3 10.sup.6 4.7 0.3 27.7 90 10.sup.9
167.7 19.4 2.8 10.sup.8 47.7 2.9 7.9 10.sup.7 8.3 0.5 13.8 10.sup.6
0.9 0.1 15.5 536 10.sup.9 413.9 91.7 4.8 10.sup.8 221.0 31.3 25.5
10.sup.7 22.3 3.8 25.8 10.sup.6 2.4 0.3 28.2 585 10.sup.9 107.7
21.0 1.2 10.sup.8 19.6 1.5 2.2 10.sup.7 9.7 1.9 10.8 10.sup.6 1.3
0.1 14.3 1482 10.sup.9 6147.2 1292.6 72.4 10.sup.8 257.7 52.6 30.3
10.sup.7 10.4 0.8 12.3 10.sup.6 1.7 0.2 19.7 1520 10.sup.9 121.2
22.3 1.5 10.sup.8 40.1 5.7 5.0 10.sup.7 16.5 5.5 20.7 10.sup.6 2.3
0.2 29.0 1583 10.sup.9 41.3 9.7 0.7 10.sup.8 12.9 1.3 2.2 10.sup.7
7.5 0.5 12.7 10.sup.6 1.1 0.1 18.7 751 10.sup.9 195.5 96.5 2.6
10.sup.8 24.7 4.9 3.2 10.sup.7 4.8 0.9 6.4 10.sup.6 0.6 0.09 8.9
931 10.sup.9 16.2 2.9 0.5 10.sup.8 4.6 0.6 1.3 10.sup.7 1.6 0.1 4.6
10.sup.6 0.2 0.01 6.7 2019 10.sup.8 67.5 1.4 1.4 10.sup.7 10.0 0.4
2.1 10.sup.6 1.1 0.1 2.4 1197 10.sup.9 20.2 3.1 0.2 10.sup.8 3.2
0.2 0.3 10.sup.7 0.5 0.2 0.4 10.sup.6 0.1 0.02 0.6 1116 10.sup.9
34.1 2.0 0.2 10.sup.8 6.3 0.2 0.4 10.sup.7 0.7 0.1 0.5 10.sup.6 0.1
0.01 0.7
[0086] The results show that the highest adhesion indices are
obtained for the following strains: a Bifidobacterium lactis ATCC
27536, a Lactobacillus johnsonii NCC 533 (CNCM I-1225) strain and
the Micrococcus varians NCC 1482 (CNCM I-1586), NCC 1520 (CNCM I-1
587) and NCC 1583 strains.
Example 2
[0087] In Vitro Assays of the Inhibition of the Adhesion of
Staphylococcus Aureus and Streptococcus pyogenes by Micrococcus
varians NCC 1482 or Lactobacillus johnsonii NCC 533.
Microorganisms and Culture Methods
[0088] The pathogens Staphylococcus aureus and Streptococcus
pyogenes are cultured in broth by subculturing from a culture in
the exponential growth phase (Table 3 ). An OD/bacterial density
correspondence was established for each of the microorganisms
assayed, on the basis of cereal dilutions and counting on agar
medium.
3TABLE 3 Bacterial strain Ref. Medium Culture conditions
Staphylococcus aureus ATCC 6538 TCS Aerobiosis, 35.degree. C./ 24 h
Streptococcus pyogenes CIP 5641 T BHI Aerobiosis, 35.degree. C./ 24
h
Culture Medium
[0089] TCS (AES, Combourg, ref. AEB 141502)
[0090] BHI (UNIPATH SA, Dardilly, ref. CM 225)
Transformed Human Keratinocytes
[0091] 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.degree. C. under 5% of CO.sub.2.
[0092] 6-well clusters (Becton Dickinson) are seeded in a
proportion of 10.sup.4 cells/cm.sup.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.
Radiolabelling
[0093] The bacteria are labelled with 2-.sup.3H-adenine (Amersham,
TRK 311), in a proportion of 100 .mu.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.
Adhesion Assay on Keratinocytes in Culture:
[0094] 1 ml of radiolabelled bacterial suspension is incubated for
1 h at 35.degree. C. The monolayer is washed 3 times with PBS
buffer and lysed by adding 1N NaOH for 30 minutes at room
temperature. The lysate is transferred into a scintillation vial
and incubated for 1 h at 60.degree. C. with 1 ml of hyamine
hydroxide (Carlo Erba, ref. 464951). The .sup.3H activity is
counted in a liquid scintillation counter. Each assay is carried
out in triplicate. A control with the plastic support is also
carried out.
[0095] The adhesion is defined by the ratio between radioactivity
which has adhered and radioactivity which was introduced,
multiplied by 100.
Adhesion Inhibition Assay
[0096] After washing and resuspension in PBS, 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.
Results
[0097] Staphylococcus aureus, Streptococcus pyogenes, Lactobacillus
johnsonii NCC 533 and Micrococcus varians NCC 1482 were assayed for
their adhesion to HaCaTen keratinocytes in culture. The results are
given in Table 4.
4TABLE 4 Microorganism Adhesion (%) 10.sup.6 cfu/ml 10.sup.7 cfu/ml
10.sup.8 cfu/ml Staphylococcus aureus -- 5.0 2.5 Streptococcus
pyogenes -- 35 42.5 NCC 533 4.5 1.7 1.2 NCC 1482 6.5 3.0 0.6
Inhibition (%) 9.0 .times. 10.sup.6 9.0 .times. 10.sup.7 9.0
.times. 10.sup.8 cfu/ml cfu/ml cfu/ml S. aureus 10.sup.8 cfu/ml +
NCC 1482 5 11 66 S. aureus 10.sup.8 cfu/ml + NCC 533 20 24 34 S.
pyogenes 10.sup.8 cfu/ml + NCC 1482 26 28 40 S. pyogenes 10.sup.8
cfu/ml + NCC 533 12 19 52
[0098] The results show that Staphylococcus aureus, Streptococcus
pyogenes, Lactobacillus johnsonii NCC 533 and Micrococcus varians
NCC 1482 adhere to the keratinocytes in culture. When the density
of the bacterial agent increases, the pathogen is increasingly
displaced.
Example 3
In Vitro Assays of the Inhibition of the Adhesion of S. aureus by
Active or Deactivated Lactobacillus johnsonii NCC 533.
[0099] 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).
[0100] 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.
Keratinocytes
[0101] For this, the HaCaT cells are cultured in DMEM supplemented
with 10% of foetal calf serum, at 37.degree. C. under 5% of
CO.sub.2. They are seeded in 6-well clusters in a proportion of
10.sup.4 cells/cm.sup.2. The adhesion assay is carried out 5 days
after confluence. The monolayers are washed 3 times with PBS before
incubation with the microorganisms.
Microorganisms
[0102] Staphylococcus aureus (ATCC 6538) is cultured in TCS medium,
in aerobiosis at 35.degree. C.
[0103] Lactobacillus johnsonii NCC 533 is cultured in MRS medium,
in anaerobiosis at 37.degree. C. For the adhesion inhibition assay,
a concentrated suspension of the bacterium is prepared in PBS
buffer, from a 48-hour culture. The suspension is adjusted to
2.times.10.sup.8 cfu/ml (OD at 525 nm=1.5). Serial dilutions are
prepared in PBS buffer in order to obtain suspensions at
2.0.times.10.sup.7 and 2.0.times.10.sup.6 cfu/ml. The various
suspensions are counted on MRS agar incubated in anaerobiosis at
37.degree. C.
[0104] 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.times.10.sup.10 cfu/g.
Radiolabelling
[0105] The radiolabelling of Staphylococcus aureus is obtained by
incorporating 100 .mu.Ci/10 ml of .sup.3H adenine during 24 h of
culturing in TCS broth. The suspension is then centrifuged for 10
minutes at 3000 rpm and washed 3 times in PBS. The cell density is
adjusted with PBS buffer to approximately 2.0.times.10.sup.8 cfu/ml
(OD at 525 nm=0.5). The specific radioactivity is determined by
scintillation counting on 100 .mu.l of the suspension.
Adhesion Inhibition Assay
[0106] 1 ml of radiolabelled suspension of Staphylococcus aureus
and 1 ml of suspension of NCC 533, per well of HaCaT cell culture,
are simultaneously added. After 1 h of incubation at 37.degree. C.,
the monolayers are washed 3 times with PBS buffer and lysed by
adding 1 ml of 1N NaOH for 30 minutes at room temperature. The
lysate is transferred into scintillation vials and incubated for 1
h at 60.degree. C. with 1 ml of benzethonium hydroxide. After
cooling, 10 ml of Hyonic fluor scintillation liquid are added, and
the radioactivity is counted on a liquid scintillation counter. The
control is obtained by adding 1 ml of radiolabelled suspension of
Staphylococcus aureus and 1 ml of PBS buffer, and corresponds to
100% of adhesion.
[0107] The results are given in Table 5.
5TABLE 5 Inhibition of the adhesion of Staphylococcus aureus to the
HaCaT cells by NCC 533 in its viable form and in its deactivated
form. Adhesion (%) Standard deviation Control 100 9 NCC 533 3.0
.times. 10.sup.6 cfu/ml 74 19 NCC 533 3.0 .times. 10.sup.7 cfu/ml
69 8 NCC 533 3.0 .times. 10.sup.8 cfu/ml 34 1 Deactivated NCC 533
3.0 .times. 10.sup.6 cfu/ml 27 2 Deactivated NCC 533 3.0 .times.
10.sup.7 cfu/ml 12 1 Deactivated NCC 533 3.0 .times. 10.sup.8
cfu/ml 6 0
Example 4
In Vivo Assays of the Inhibition of the Adhesion of Skin Pathogens
by Deactivated Lactobacillus johnsonii
Materials and Methods
[0108] Animals: 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.
[0109] 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.
Preparation of the Inoculum
[0110] A suspension of the bacterium is prepared for inoculation in
the mice. For this, a preculture in the exponential growth phase of
strain 1 is prepared on a solid medium (AES, AEB 122 859) at
35.degree. C. for 18 to 24 h. After incubation, 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.sup.8 cfu/ml.
Inoculation of the Mice
[0111] The skin of the mice is delipidized on the flanks with 95
[sic] ethanol (Merck). 50 .mu.l of a suspension containing a 50/50
mixture of the S. aureus inoculum, 10.sup.7 cfu/ml, and of the
product to be assayed were slowly applied to the delipidized area
(6.25 cm.sup.2), using a micropipette. The inoculated sites are
protected by occlusion for 1 h under a sterile plastic dressing
(Dermafilm 33.times.15, ref. 38.3015, Vygon laboratory).
Counting of the Viable Bacteria of the Lesions
[0112] 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.).
[0113] 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.degree. C., the colonies
developed are counted and the CFU(colony forming units) are
determined.
[0114] Results: The results are given in Table 6.
6TABLE 6 1% NCC 533 assay Log cfu/cm.sup.2 S.aureus + PBS 3.4
S.aureus + 1% NCC 533 in PBS 2.6 * S.aureus + 0.045% glutaraldehyde
0.8 * * significant with respect to the control (SA/PBS), Student's
T test (n = 5)
[0115] The presence of NCC 533 at 1% makes it possible to decrease
the number of bacteria found by approximately 1 log after 4 hours
of contact. The difference appears to be significant with respect
to the control (p=0.098).
[0116] In the presence of glutaraldehyde, the decrease in the
number of bacteria is even more significant with respect to the
control, however it is not out of the question that this activity
is due to its antiseptic activity and acts as soon as the S. aureus
is added to the mixture; thus, the activity observed after 4 h
would not be due to an anti-adhesive effect, but to an
antibacterial activity of the product.
[0117] The results are given in Table 7.
7TABLE 7 % of Inoculum decrease Significance size cfu/ml Treatment
vs. control vs. control 10.sup.6 cfu/ml 0.5% NCC 533 20.1% P =
0.019 (*) 1% NCC 533 22.6% P = 0.016 (*) 0.045% Glutaraldehyde
87.9% P = 0.0001 (***) 10.sup.7 cfu/ml 0.5% NCC 533 27.4% P =
0.0015 (*) 1% NCC 533 19.9% P = 0.0004 (***) 0.045% Glutaraldehyde
92.0% P = 0.0004 (***) * p < 0.05, *** p < 0.001 significant
with respect to the control (SA/PBS)
[0118] The presence of NCC 533 at 0.5% and 1% decreases the number
of S. aureus bacteria found by approximately 1 log for the inocula
at 10.sup.6 cfu/ml and 10.sup.7 cfu/ml. No dose effect is observed,
either with the 10.sup.6 cfu/ml inoculum or with the inoculum at
10.sup.7 cfu/ml.
[0119] In the presence of glutaraldehyde at 0.045%, the decrease is
much greater with respect to the control; it is approximately 3
logs.
[0120] These results confirm the activity of the deactivated NCC
533 at 0.5% and 1% as an inhibitor of the adhesion of S.
aureus.
Example 5
Body Lotion
[0121] 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.times.10.sup.8 to
10.times.10.sup.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.degree. C. for about 2 hours. The mixture is made up to 100%
with water.
[0122] 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.
Example 6
Shampoo
[0123] 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.sup.8 to 10.sup.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.degree. C. for about 2 hours. The mixture is made
up to 100% with water.
[0124] The shampoo thus prepared has properties which regulate
scalp pathogenic flora. It is in particular indicated in the
treatment of dandruff.
Example 7
[0125] In order to obtain a pharmaceutical composition with
properties which regulate skin pathogenic flora, fatty and aqueous
phases are prepared which have the following composition:
8 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%
Propyl POB 0.05% Aqueous phase: Water Qs 100% Glycerol 5% Methyl
POB 0.1%
[0126] The fatty and aqueous phases are heated to 75.degree. 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 8
[0127] In the same way as in Example 7, a composition is prepared
which has the following composition:
9 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 9
[0128] In the same way as in Example 7, a composition is prepared
which has the following composition:
10 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%
Example 10
Shampoo for Pets
[0129] 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.
[0130] The shampoo thus prepared has properties which regulate the
pathogenic flora of the cutaneous system of pets.
* * * * *