U.S. patent application number 14/860836 was filed with the patent office on 2016-01-14 for probiotic bacteria for the topical treatment of skin disorders.
This patent application is currently assigned to DUPONT NUTRITION BIOSCIENCES APS. The applicant listed for this patent is DUPONT NUTRITION BIOSCIENCES APS. Invention is credited to Arthur Ouwehand, Heli Putaala, Nina Rautonen, Kirsti Tihonen.
Application Number | 20160008412 14/860836 |
Document ID | / |
Family ID | 46395577 |
Filed Date | 2016-01-14 |
United States Patent
Application |
20160008412 |
Kind Code |
A1 |
Putaala; Heli ; et
al. |
January 14, 2016 |
PROBIOTIC BACTERIA FOR THE TOPICAL TREATMENT OF SKIN DISORDERS
Abstract
The present invention relates to a probiotic bacteria, and/or
soluble metabolite of a probiotic bacteria and/or a cell lysate of
a probiotic bacteria for use in the treatment of a disorder
associated with Tight Junction function, characterised in that the
probiotic bacteria, soluble metabolite of a probiotic bacteria
and/or a cell lysate of a probiotic bacteria is formulated for
topical administration.
Inventors: |
Putaala; Heli; (Kirkkonummi,
FI) ; Ouwehand; Arthur; (Inga, FI) ; Tihonen;
Kirsti; (Helsinki, FI) ; Rautonen; Nina;
(Espoo, FI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DUPONT NUTRITION BIOSCIENCES APS |
Copenhagen |
|
DK |
|
|
Assignee: |
DUPONT NUTRITION BIOSCIENCES
APS
Copenhagen
DK
|
Family ID: |
46395577 |
Appl. No.: |
14/860836 |
Filed: |
September 22, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14114591 |
Oct 29, 2013 |
|
|
|
PCT/EP2012/058047 |
May 2, 2012 |
|
|
|
14860836 |
|
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Current U.S.
Class: |
424/450 ;
424/780; 424/93.4; 424/93.45 |
Current CPC
Class: |
A61P 37/00 20180101;
A61K 35/747 20130101; A61P 17/00 20180101; A61P 17/06 20180101;
A61P 17/16 20180101; A61K 35/744 20130101; A61K 31/205 20130101;
A61K 35/741 20130101; A61P 17/18 20180101; A61K 35/745
20130101 |
International
Class: |
A61K 35/741 20060101
A61K035/741; A61K 35/745 20060101 A61K035/745; A61K 31/205 20060101
A61K031/205; A61K 35/747 20060101 A61K035/747 |
Foreign Application Data
Date |
Code |
Application Number |
May 3, 2011 |
EP |
11164534.7 |
Claims
1-16. (canceled)
17. A method for treating a disorder associated with Tight Junction
function, said method comprising topical administration of a
formulation comprising a probiotic bacteria, soluble metabolite of
a probiotic bacteria and/or a cell lysate of a probiotic bacteria
to a body surface in need of treatment for said disorder.
18. The method of claim 17, wherein said formulation is topically
administered to the skin.
19. The method of claim 17, wherein said formulation is topically
administered to a mucous membrane, wherein said mucous membrane is
selected from the group consisting of the mucosa of: the vagina,
the penis, the uretha, the bladder, the anus, the mouth, the nose,
the throat, the bronchi, the lungs, the eye and the ear.
20. The method according to claim 17, wherein the formulation
comprises more than one probiotic bacteria and/or soluble
metabolite and/or cell lysates.
21. The method according to claim 17, wherein the bacteria
comprises at least one lactic acid bacteria.
22. The method according to claim 17, wherein the probiotic
bacteria is selected from the group consisting of one or more of
Lactobacillus acidophilus, Lactobacillus salivarius,
Bifidobacterium lactis and Propionibacterium jensenii.
23. The method according to claim 17, wherein the formulation
comprises said probacteria and/or said cell lysate of a probiotic
bacteria, wherein said probiotic bacteria is selected from the
group consisting of one or more of Lactobacillus acidophilus NCFM,
Lactobacillus salivarius Ls-33, and Propionibacterium jensenii
P63.
24. The method according to claim 17, wherein the formulation
comprises said soluble metabolite of a probacteria, and wherein
said probiotic bacteria is selected from the group consisting of
one or more of Lactobacillus acidophilus NCFM, Lactobacillus
salivarius Ls-33 and Bifidobacterium lactis 420.
25. The method of claim 24, wherein the probiotic bacteria is
Bifidobacterium lactis 420.
26. The method according to claim 17, wherein the formulation
comprises said cell lysate of a probiotic bacteria and/or said
metabolite of a probacteria, and wherein said probiotic bacteria is
L. acidophilus La-14.
27. The method according to claim 17, wherein said formulation
further comprises at least one of betaine, a polyol (e.g. xylitol
or lactitol) and/or a polyphenol (e.g. epicatechin or
gallocatechin).
28. The method according to claim 17, wherein the disorder
associated with Tight Junction function is selected from the group
consisting of psoriasis, acne, atopic dermatitis dry skin, allergy,
rashes, UV-irritated skin, detergent irritated skin (including
irritation caused by enzymes used in washing detergents and sodium
lauryl sulphate) and thinning skin (e.g. skin from the elderly and
children).
29. The method according to claim 17, wherein the formulation
further comprises a pharmaceutically acceptable carrier.
30. The method according to claim 17, wherein the formulation
comprises between about 0.01% and about 30% by weight of probiotic
bacteria, soluble metabolite and/or cell lysate.
31. The method according to claim 17, wherein the formulation is
selected from the group consisting of a cream, lotion, spray,
solution, gel, ointment, paste, plaster, paint, bioadhesive or
suspension.
32. The method according to claim 30, wherein the formulation
comprises liposomes, micelles, and/or microspheres.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/114,591, which is the U.S. National Stage of International
Application PCT/EP2012/058047 filed May 2, 2012, which designates
the U.S. and was published by the International Bureau in English
on Nov. 8, 2012, and which claims the benefit of European
Application No. 11164534.7, filed May 3, 2011, both of which are
hereby incorporated by reference in their entirety.
FIELD OF INVENTION
[0002] The present invention relates to probiotic bacteria and/or
soluble metabolites of a probiotic bacteria and/or a cell lysate of
a probiotic bacteria for use in the topical treatment of a disorder
associated with skin barrier function.
BACKGROUND OF THE INVENTION
[0003] The epidermis, the squamous stratified epithelium of the
skin, consists of multiple sublayers and is one of the most
important barriers of the body with the outside world. Stratum
corneum is the outermost layer of the epidermis and the final
anucleated step in keratinocyte differentiation from the cells in
nucleated epidermal layers. Although stratum corneum is recognised
as the most important physical barrier, the nucleated epidermal
layers are also significant in barrier function as evidenced by
studies with burn victims (1, 2). Together, the skin barrier
protects against extensive water loss in one direction
(inside-outside barrier) and against the invasion of harmful
substances from the environment (outside-inside barrier) (2). The
maintenance of the barrier is also important for balanced
proliferation in the basal layer and preservation of the calcium
ion gradient and thus proper epidermal differentiation (3).
[0004] In the mammalian epidermis, tight junctions (TJ) are mainly
found in the stratum granulosum, or the granular layer of the
epithelium, located underneath the stratum corneum (4). TJ are
dynamic cell-cell junctions that connect neighbouring cells,
control the paracellular pathway of molecules acting as barriers,
and have a fence function by separating apical cell membranes from
basolateral cell membranes (5). They are important not only in
inside-outside barrier function but also in outside-inside barrier
function.
[0005] Probiotics have been defined as "live microorganisms, which
when administered in adequate amounts, confer health benefit on the
host" (13). Research of the effects of oral administration of
probiotics on the epidermis has previously been undertaken.
Probiotics have been suggested to modulate the immunity
systemically and have been suggested for prophylactic use to
alleviate atopic eczema in children (17).
[0006] Few studies have been undertaken to study how probiotics
affect keratinocytes when administered topically. However, in one
study, sonicated lysates of Streptococcus thermophilus have been
observed to increase the level of ceramides in stratum corneum in
vitro and in vivo (22).
SUMMARY OF THE INVENTION
[0007] The present invention is based on the inventors surprising
discovery that topical administration of probiotic bacteria, and/or
soluble metabolites of probiotic bacteria and/or a cell lysates of
probiotic bacteria can improve Tight Junction (TJ) function in the
epithelium.
[0008] The importance of the role of TJ as part of the epidermal
barrier function of the skin has been verified by discovering that
deficiency of the TJ protein claudin 1 (cldn-1) results in fatal
epidermal water-loss in neonate mice (4). In various skin diseases
with perturbed barrier function, such as psoriasis, zonula
occludens protein 1 (ZO-1) and occludin are relocalised to the
lower epidermal layers (6). TJ proteins are also known to be
targets for bacterial, and viral insults, and certain viruses and
bacteria use them as receptors in pathogenesis (8). Moreover,
allergens may disrupt TJ and promote asthma (9). In addition,
UV-induced epidermal barrier perturbation is associated with TJ
barrier impairment (10). Therefore, TJ are important not only in
inside-outside barrier but also in outside-inside barrier.
[0009] Therefore, according to a first broad aspect of the present
invention there is provided a probiotic bacteria, and/or soluble
metabolites of a probiotic bacteria and/or a cell lysate of a
probiotic bacteria for use in the treatment of a disorder
associated with Tight Junction function, characterised in that the
probiotic bacteria, soluble metabolite of a probiotic bacteria
and/or a cell lysate of a probiotic bacteria is formulated for
topical administration.
[0010] According to a second aspect of the present invention there
is provided a method of treating a patient suffering from a
disorder associated with Tight Junction function comprising
topically administering to a patient a probiotic bacteria, and/or
soluble metabolites of a probiotic bacteria and/or a cell lysate of
a probiotic bacteria.
[0011] According to a third aspect of the present invention there
is provided the use of a probiotic bacteria, and/or soluble
metabolites of a probiotic bacteria and/or a cell lysate of a
probiotic bacteria in the manufacture of a topical medicament for
use in the treatment of a disorder associated with Tight Junction
function.
DETAILED DESCRIPTION OF THE INVENTION
[0012] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs.
Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR
BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale
& Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper
Perennial, NY (1991) provide one of skill with a general dictionary
of many of the terms used in this disclosure.
[0013] This disclosure is not limited by the exemplary methods and
materials disclosed herein, and any methods and materials similar
or equivalent to those described herein can be used in the practice
or testing of embodiments of this disclosure. Numeric ranges are
inclusive of the numbers defining the range.
[0014] Other definitions of terms may appear throughout the
specification. Before the exemplary embodiments are described in
more detail, it is to understand that this disclosure is not
limited to particular embodiments described. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be
limiting, since the scope of the present disclosure will be limited
only by the appended claims.
[0015] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within this disclosure. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within this disclosure, subject to any specifically excluded limit
in the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in this disclosure.
[0016] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise.
[0017] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
such publications constitute prior art to the claims appended
hereto.
[0018] It will be understood that in the following, preferred
embodiments referred to in relation to one broad aspect of the
invention are equally applicable to each of the other broad aspects
of the present invention described above. It will be further
understood that, unless the context dictates otherwise, the
preferred embodiments described below may be combined.
[0019] When used herein, the term topical includes references to
formulations that are adapted for application to body surfaces
(e.g. the skin or mucous membranes). Mucous membranes that may be
mentioned in this respect include the mucosa of the vagina, the
penis, the urethra, the bladder, the anus, the mouth (including the
mucosa of the cheek, the soft palate, the under surface of tongue
and the floor of the mouth), the nose, the throat (including the
mucosa of the pharynx, the larynx, the trachea and the esophagus),
the bronchi, the lungs, the eye and the ear.
[0020] In preferred embodiments of the present invention, the
probiotic bacteria, soluble metabolite and/or cell lysate is
formulated for administration to the skin.
[0021] It will be further understood that the formulation for use
in the present invention may comprise one or more of at least one
probiotic bacteria, at least one soluble metabolite of a probiotic
bacteria and/or at least one cell lysate of a probiotic
bacteria.
[0022] It will be further understood that the formulation may
comprise more than one probiotic bacteria, soluble metabolite
and/or cell lysate. For example the formulation may comprise at
least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 or 20 probiotic bacteria
or their soluble metabolites or cell lysates.
[0023] It will be understood by the skilled person that as used
herein the term probiotic refers to a live microorganism (including
bacteria or yeasts for example) which, when topically applied in
sufficient numbers, beneficially affects the host organism, i.e. by
conferring one or more demonstrable health benefits on the host
organism.
[0024] It will also be readily apparent that the formulations of
the present invention may further include one or more
prebiotics.
[0025] Whilst there are no lower or upper limits for probiotic use,
it has been suggested that at least 10.sup.6-10.sup.12, preferably
at least 10.sup.6-10.sup.10, preferably 10.sup.8-10.sup.9, cfu as a
daily dose will be effective to achieve the beneficial health
effects in a subject.
[0026] As used herein, the term "soluble metabolite" refers to a
metabolite or metabolites present in the supernatant of a cell
culture from which the cells have been removed. In preferred
embodiments the culture is grown to a cell density of at least
about OD.sub.600 0.5. In a further preferred embodiment the cells
are removed by centrifugation. In a more preferred embodiment the
supernatant is filtered. It will be apparent that the supernatant
may be used directly in the formulations of the present invention,
or that one or more of the metabolites may be isolated form the
supernatant by any suitable means prior to use.
[0027] As used herein, the term "cell lysate" or "lysate" refers to
probiotic cells which have been lysed by any suitable means. In
preferred embodiments, the cell debris is removed prior to use. In
more preferred embodiments the cell lysates are filtered prior to
use. In exemplary embodiments, the cells are lysed by, for example
sonication, homogenisation, shearing or chemical lysis.
[0028] Probiotic bacteria suitable for use in the present invention
include, but are not limited to, Bifidobacterium, Brevibacterium,
Propionibacterium, Lactococcus, Streptococcus, Lactobacillus (e.g.,
L. acidophilus), Enterococcus, Pediococcus, Leuconostoc, and/or
Oenococcus.
[0029] Soluble metabolites for use in the present invention
include, but are not limited to, soluble metabolites from
Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus,
Streptococcus, Lactobacillus (e.g., L. acidophilus), Enterococcus,
Pediococcus, Leuconostoc, and/or Oenococcus.
[0030] Cell lysates for use in the present invention include, but
are not limited to, cell lysates from Bifidobacterium,
Brevibacterium, Propionibacterium, Lactococcus, Streptococcus,
Lactobacillus (e.g., L. acidophilus), Enterococcus, Pediococcus,
Leuconostoc, and/or Oenococcus.
[0031] Preferably, the probiotic bacteria, soluble metabolite
and/or cell lysate for use according to the present invention
comprises at least one lactic acid bacteria.
[0032] More preferably, the probiotic bacteria, soluble metabolite
and/or cell lysate is selected from the group comprising
Lactobacillus acidophilus, Lactobacillus salivarius,
Bifidobacterium lactis and Propionibacterium jensenii.
[0033] In an even more preferred embodiment the bacteria or cell
lysate is selected from Lactobacillus acidophilus NCFM,
Lactobacillus salivarius Ls-33 and P. jensenii P63.
[0034] In an alternative preferred embodiment the soluble
metabolite of a probiotic bacteria is from Bifidobacterium lactis
420.
[0035] In one preferred embodiment of the present invention, the
formulation further comprises at least one of betaine, a polyol
(e.g. xylitol or lactitol) and/or a polyphenol (e.g. epicatechin or
gallocatechin).
[0036] It will be understood that the disorder may be any disorder
associated with Tight Junction function. In preferred embodiments
the disorder is selected from the group comprising psoriasis, acne,
atopic dermatitis, dry skin, allergy, rashes, UV-irritated skin,
detergent irritated skin (including irritation caused by enzymes
used in washing detergents and sodium lauryl sulphate), thinning
skin (e.g. skin from the elderly and children).
[0037] It will be further apparent that the formulation for use
according to the present invention may comprise any
pharmaceutically effective amount of the probiotic bacteria,
soluble metabolite and/or cell lysate, for example, at least about
0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%,
about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about
1.0%, about 1.5%, about 2.0%, about 3.0%, about 4.0%, about 5.0%,
about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0%, about
11.0%, about 12.0%, about 13.0%, about 14.0%, about 15.0%, about
16.0%, about 17.0%, about 18.0%, about 19.0%, about 20.0%, about
25.0%, about 30.0%, about 35.0%, about 40.0%, about 45.0%, about
50.0% by weight of probiotic bacteria, soluble metabolite and/or
cell lysate.
[0038] In an alternative embodiment the formulation for use
according to the present invention may comprise, for example, at
least about 0.01% to about 30%, about 0.01% to about 20%, about
0.01% to about 5%, about 0.1% to about 30%, about 0.1% to about
20%, about 0.1% to about 15%, about 0.1% to about 10%, about 0.1%
to about 5%, about 0.2% to about 5%, about 0.3% to about 5%, about
0.4% to about 5%, about 0.5% to about 5%, about 1% to about 5%, by
weight of probiotic bacteria, soluble metabolite and/or cell
lysate.
[0039] The topical formulation for use in the present invention may
be in any form suitable for application to the body surface, such
as a cream, lotion, sprays, solution, gel, ointment, paste,
plaster, paint, bioadhesive, suspensions or the like, and/or may be
prepared so as to contain liposomes, micelles, and/or microspheres.
Such a formulation may be used in combination with an occlusive
overlayer so that moisture evaporating from the body surface is
maintained within the formulation upon application to the body
surface and thereafter.
[0040] Topical formulations include those in which the active
ingredient(s) is (are) dissolved or dispersed in a dermatological
vehicle known in the art (e.g. aqueous or non-aqueous gels,
ointments, water-in-oil or oil-in-water emulsions). Constituents of
such vehicles may comprise water, aqueous buffer solutions,
non-aqueous solvents (such as ethanol, isopropanol, benzyl alcohol,
2-(2-ethoxyethoxy)ethanol, propylene glycol, propylene glycol
monolaurate, glycofurol or glycerol), oils (e.g. a mineral oil such
as a liquid paraffin, natural or synthetic triglycerides such as
Miglyol.TM., or silicone oils such as dimethicone). Depending,
inter alia, upon the nature of the formulation as well as its
intended use and site of application, the dermatological vehicle
employed may contain one or more components (for example, when the
formulation is an aqueous gel, components in addition to water)
selected from the following list:
[0041] a solubilising agent or solvent (e.g. a .beta.-cyclodextrin,
such as hydroxypropyl .beta.-cyclodextrin, or an alcohol or polyol
such as ethanol, propylene glycol or glycerol);
[0042] a thickening agent (e.g. hydroxyethylcellulose,
hydroxypropylcellulose, carboxymethylcellulose or carbomer);
[0043] a gelling agent (e.g. a polyoxyethylene-polyoxypropylene
copolymer);
[0044] a preservative (e.g. benzyl alcohol, benzalkonium chloride,
chlorhexidine, chlorbutol, a benzoate, potassium sorbate or EDTA or
salt thereof); and
[0045] pH buffering agent(s) (such as a mixture of dihydrogen
phosphate and hydrogen phosphate salts, or a mixture of citric acid
and a hydrogen phosphate salt).
[0046] In particular compositions, [0047] (i) water may be present
at from 55 to 75% (e.g. from 60 to 72.5%) by weight; [0048] (ii)
the one or more polar, non-aqueous solvents may (together) be
present at from 15 to 40% (e.g. from 24 to 35%) by weight; [0049]
(iii) glycerol, if used, may be present at from 5 to 25% (e.g. from
15 to 20%) by weight; [0050] (iv) ethanol, if used, may be present
at from 3 to 10% (e.g. from 5 to 8%) by weight; [0051] (v)
propylene glycol, if used, may be present at from 2 to 15% (e.g.
from 4 to 6%) by weight; [0052] (vi) the preservative may be
present at from 0.1 to 3% (e.g. about 1%) by weight; [0053] (vii)
the thickening agent may be present at from 1 to 5% (e.g. about 2%
by weight).
[0054] In further particular topical compositions, the pH buffering
agent(s) may, if employed and when dissolved in the water component
of the composition, provide a pH in the range of 5 to 7 (e.g. about
pH 5.5).
[0055] Methods of producing topical pharmaceutical compositions
such as creams, ointments, lotions, sprays and sterile aqueous
solutions or suspensions are well known in the art. Suitable
methods of preparing topical pharmaceutical compositions are
described, for example in WO 95/10999, U.S. Pat. No. 6,974,585, WO
2006/048747 (incorporated herein by reference), as well as in
documents cited in any of these references.
[0056] A pharmaceutically acceptable carrier may also be
incorporated in the formulation of the present invention and may be
any carrier conventionally used in the art. Examples thereof
include water, lower alcohols, higher alcohols, polyhydric
alcohols, monosaccharides, disaccharides, polysaccharides,
hydrocarbon oils, fats and oils, waxes, fatty acids, silicone oils,
nonionic surfactants, ionic surfactants, silicone surfactants, and
water-based mixtures and emulsion-based mixtures of such
carriers.
[0057] The term "pharmaceutically acceptable" or "pharmaceutically
acceptable carrier" is used herein to refer to a compound or
composition that may be incorporated into a pharmaceutical
formulation without causing undesirable biological effects or
unwanted interaction with other components of the formulation.
[0058] "Carriers" or "vehicles" as used herein refer to carrier
materials suitable for incorporation in a topically applied
composition. Carriers and vehicles useful herein include any such
materials known in the art, which are non toxic and do not interact
with other components of the formulation in which it is contained
in a deleterious manner.
[0059] The term "aqueous" refers to a formulation that contains
water or that becomes water-containing following application to the
skin or mucosal tissue.
[0060] The pharmaceutical formulation of the invention comprises a
pharmaceutically acceptable topical carrier and an active agent
that consists essentially of a probiotic bacteria and/or
metabolites of a probiotic bacteria and or a cell lysate of a
probiotic bacteria.
[0061] Formulations of the invention may optionally contain a
pharmaceutically acceptable viscosity enhancer and/or film former.
A viscosity enhancer increases the viscosity of the formulation so
as to inhibit its spread beyond the site of application. Balsam Fir
(Oregon) is an example of a pharmaceutically acceptable viscosity
enhancer.
[0062] A film former, when it dries, forms a protective film over
the site of application. The film inhibits removal of the active
ingredient and keeps it in contact with the site being treated. An
example of a film former that is suitable for use in this invention
is Flexible Collodion, USP. As described in Remington: The Science
and Practice of Pharmacy, 19th Ed. (Easton, Pa.: Mack Publishing
Co., 1995), at page 1530, collodions are ethyl ether/ethanol
solutions containing pyroxylin (a nitrocellulose) that evaporate to
leave a film of pyroxylin. A film former may act additionally as a
carrier. Solutions that dry to form a film are sometimes referred
to as paints.
[0063] Creams, as is well known in the arts of pharmaceutical
formulation, are viscous liquids or semisolid emulsions, either
oil-in-water or water-in-oil. Cream bases are water-washable, and
contain an oil phase, an emulsifier, and an aqueous phase. The oil
phase, also called the "internal" phase, is generally comprised of
petrolatum and a fatty alcohol such as cetyl or stearyl alcohol.
The aqueous phase usually, although not necessarily, exceeds the
oil phase in volume, and generally contains a humectant. The
emulsifier in a cream formulation is generally a nonionic, anionic,
cationic or amphoteric surfactant.
[0064] Lotions, are preparations to be applied to the skin surface
without friction, and are typically liquid or semiliquid
preparations in which particles, including the active agent, are
present in a water or alcohol base. Lotions are usually suspensions
of solids, and preferably, comprise a liquid oily emulsion of the
oil-in-water type. Lotions are preferred formulations herein for
treating large body areas, because of the ease of applying a more
fluid composition. It is generally necessary that the insoluble
matter in a lotion be finely divided. Lotions will typically
contain suspending agents to produce better dispersions as well as
compounds useful for localizing and holding the active agent in
contact with the skin, e.g., methylcellulose, sodium
carboxymethyl-cellulose, or the like.
[0065] Solutions are homogeneous mixtures prepared by dissolving
one or more chemical substances (solutes) in a liquid such that the
molecules of the dissolved substance are dispersed among those of
the solvent. The solution may contain other pharmaceutically or
cosmetically acceptable chemicals to buffer, stabilize or preserve
the solute. Common examples of solvents used in preparing solutions
are ethanol, water, propylene glycol or any other acceptable
vehicles.
[0066] As is of course well known, gels are semisolid,
suspension-type systems. Single-phase gels contain organic
macromolecules distributed substantially uniformly throughout the
carrier liquid, which is typically aqueous, but also, preferably,
contain an alcohol, and, optionally, an oil. Preferred "organic
macromolecules," i.e., gelling agents, are crosslinked acrylic acid
polymers such as the "carbomer" family of polymers, e.g.,
carboxypolyalkylenes that may be obtained commercially under the
Carbopol.RTM. trademark. Also preferred are hydrophilic polymers
such as polyethylene oxides, polyoxyethylene-polyoxypropylene
copolymers and polyvinylalcohol; cellulosic polymers such as
hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl
methylcellulose, hydroxypropyl methylcellulose phthalate, and
methyl cellulose; gums such as tragacanth and xanthan gum; sodium
alginate; and gelatin. In order to prepare a uniform gel,
dispersing agents such as alcohol or glycerin can be added, or the
gelling agent can be dispersed by trituration, mechanical mixing or
stirring, or combinations thereof.
[0067] Ointments, as also well known in the art, are semisolid
preparations that are typically based on petrolatum or other
petroleum derivatives. The specific ointment base to be used, as
will be appreciated by those skilled in the art, is one that will
provide for a number of desirable characteristics, e.g., emolliency
or the like. As with other carriers or vehicles, an ointment base
should be inert, stable, nonirritating, and nonsensitizing. As
explained in Remington: The Science and Practice of Pharmacy, 19th
Ed. (Easton, Pa.: Mack Publishing Co., 1995), at pages 1399-1404,
ointment bases may be grouped in four classes: oleaginous bases;
emulsifiable bases; emulsion bases; and water-soluble bases.
Oleaginous ointment bases include, for example, vegetable oils,
fats obtained from animals, and semisolid hydrocarbons obtained
from petroleum. Emulsifiable ointment bases, also known as
absorbent ointment bases, contain little or no water and include,
for example, hydroxystearin sulfate, anhydrous lanolin, and
hydrophilic petrolatum. Emulsion ointment bases are either
water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, and
include, for example, acetyl alcohol, glyceryl monostearate,
lanolin, and stearic acid. Preferred water-soluble ointment bases
are prepared from polyethylene glycols of varying molecular weight;
again, see Remington: The Science and Practice of Pharmacy for
further information.
[0068] Pastes are semisolid dosage forms in which the active agent
is suspended in a suitable base. Depending on the nature of the
base, pastes are divided between fatty pastes or those made from
single-phase aqueous gels. The base in a fatty paste is generally
petrolatum or hydrophilic petrolatum or the like. The pastes made
from single-phase aqueous gels generally incorporate
carboxymethylcellulose or the like as a base.
[0069] Formulations may also be prepared with liposomes, micelles,
and microspheres. Liposomes are microscopic vesicles having a lipid
wall comprising a lipid bilayer, and, in the present context,
encapsulate one or more components of the formulations. Liposomal
preparations herein include cationic (positively charged), anionic
(negatively charged), and neutral preparations. Cationic liposomes
are readily available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethyl-ammonium (DOTMA)
liposomes are available under the tradename Lipofectin.RTM. (GIBCO
BRL, Grand Island, N.Y.). Similarly, anionic and neutral liposomes
are readily available as well, e.g., from Avanti Polar Lipids
(Birmingham, Ala.), or can be easily prepared using readily
available materials. Such materials include phosphatidyl choline,
cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl
choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and
dioleoylphoshatidyl ethanolamine (DOPE), among others. These
materials can also be mixed with DOTMA in appropriate ratios.
Methods for making liposomes using these materials are well known
in the art.
[0070] Micelles are known in the art as comprised of surfactant
molecules arranged so that their polar headgroups form an outer
spherical shell, while the hydrophobic, hydrocarbon chains are
oriented towards the center of the sphere, forming a core. Micelles
form in an aqueous solution containing surfactant at a high enough
concentration so that micelles naturally result. Surfactants useful
for forming micelles include, but are not limited to, potassium
laurate, sodium octane sulfonate, sodium decane sulfonate, sodium
dodecane sulfonate, sodium lauryl sulfate, docusate sodium,
decyltrimethylammonium bromide, dodecyltrimethylammonium bromide,
tetradecyltrimethylammonium bromide, tetradecyltrimethyl-ammonium
chloride, dodecylammonium chloride, polyoxyl-8 dodecyl ether,
polyoxyl-12 dodecyl ether, nonoxynol 10, and nonoxynol 30.
[0071] Microspheres, similarly, may be incorporated into the
present formulations. Like liposomes and micelles, microspheres
essentially encapsulate one or more components of the present
formulations. They are generally although not necessarily formed
from lipids, preferably charged lipids such as phospholipids.
Preparation of lipidic microspheres is well known in the art and
described in the pertinent texts and literature.
[0072] Various additives, known to those skilled in the art, may be
included in the topical formulations. For example, solvents,
including relatively small amounts of alcohol, may be used to
solubilize certain formulation components. With particularly severe
skin conditions it may be desirable to include an added permeation
enhancer in the formulation. Examples of suitable enhancers
include, but are not limited to, ethers such as diethylene glycol
monoethyl ether (available commercially as Transcutol.RTM.) and
diethylene glycol monomethyl ether; surfactants such as sodium
laurate, sodium lauryl sulfate, cetyltrimethylammonium bromide,
benzalkonium chloride, Poloxamer.RTM. (231, 182, 184), Tween.RTM.
(20, 40, 60, 80), and lecithin (U.S. Pat. No. 4,783,450); alcohols
such as ethanol, propanol, octanol, benzyl alcohol, and the like;
polyethylene glycol and esters thereof such as polyethylene glycol
monolaurate (PEGML; see, e.g., U.S. Pat. No. 4,568,343); amides and
other nitrogenous compounds such as urea, dimethylacetamide (DMA),
dimethylformamide (DMF), 2-pyrrolidone, 1-methyl-2-pyrrolidone,
ethanolamine, diethanolamine, and triethanolamine; terpenes;
alkanones; and organic acids, particularly citric acid and succinic
acid. Azone.RTM. and sulfoxides such as DMSO and C.sub.10MSO may
also be used, but are less preferred.
[0073] Most preferred enhancers are those lipophilic co-enhancers
typically referred to as "plasticizing" enhancers, i.e., enhancers
that have a molecular weight in the range of about 150 to 1000, an
aqueous solubility of less than about 1 wt. %, preferably less than
about 0.5 wt. %, and most preferably less than about 0.2 wt. %. The
Hildebrand solubility parameter .delta. of plasticizing enhancers
is in the range of about 2.5 to about 10, preferably in the range
of about 5 to about 10. Preferred lipophilic enhancers are fatty
esters, fatty alcohols, and fatty ethers. Examples of specific and
most preferred fatty acid esters include methyl laurate, ethyl
oleate, propylene glycol monolaurate, propylene glycerol dilaurate,
glycerol monolaurate, glycerol monooleate, isopropyl n-decanoate,
and octyldodecyl myristate. Fatty alcohols include, for example,
stearyl alcohol and oleyl alcohol, while fatty ethers include
compounds wherein a diol or triol, preferably a C.sub.2-C.sub.4
alkane diol or triol, are substituted with one or two fatty ether
substituents.
[0074] Additional permeation enhancers will be known to those of
ordinary skill in the art of topical drug delivery, and/or are
described in the pertinent texts and literature. See, e.g.,
Percutaneous Penetration Enhancers, eds. Smith et al. (CRC Press,
1995).
[0075] Various other additives may be included in the compositions
of the present invention in addition to those identified above.
These include, but are not limited to, antioxidants, astringents,
perfumes, preservatives, emollients, pigments, dyes, humectants,
propellants, and sunscreen agents, as well as other classes of
materials whose presence may be pharmaceutically or otherwise
desirable. Typical examples of optional additives for inclusion in
the formulations of the invention are as follows: preservatives
such as sorbate; solvents such as isopropanol and propylene glycol;
astringents such as menthol and ethanol; emollients such as
polyalkylene methyl glucosides; humectants such as glycerine;
emulsifiers such as glycerol stearate, PEG-100 stearate,
polyglyceryl-3 hydroxylauryl ether, and polysorbate 60; sorbitol
and other polyhydroxyalcohols such as polyethylene glycol;
sunscreen agents such as octyl methoxyl cinnamate (available
commercially as Parsol MCX) and butyl methoxy benzoylmethane
(available under the tradename Parsol 1789); antioxidants such as
ascorbic acid (vitamin C), .alpha.-tocopherol (Vitamin E),
.beta.-tocopherol, .gamma.-tocopherol, .delta.-tocopherol,
.epsilon.-tocopherol, .zeta..sub.1-tocopherol,
.zeta..sup.2-tocopheroI, .eta.-tocopherol, and retinol (vitamin A);
essential oils, ceramides, essential fatty acids, mineral oils,
vegetable oils (e.g., soya bean oil, palm oil, liquid fraction of
shea butter, sunflower oil), animal oils (e.g., perhydrosqualene),
synthetic oils, silicone oils or waxes (e.g., cyclomethicone and
dimethicone), fluorinated oils (generally perfluoropolyethers),
fatty alcohols (e.g., cetyl alcohol), and waxes (e.g., beeswax,
carnauba wax, and paraffin wax); skin-feel modifiers; and
thickeners and structurants such as swelling clays and cross-linked
carboxypolyalkylenes that may be obtained commercially under the
Carbopol.RTM. trademark.
[0076] Other additives include beneficial agents such as those
materials that condition the skin (particularly, the upper layers
of the skin in the stratum corneum) and keep it soft by retarding
the decrease of its water content and/or protect the skin. Such
conditioners and moisturizing agents include, by way of example,
pyrrolidine carboxylic acid and amino acids; organic antimicrobial
agents such as 2,4,4'-trichloro-2-hydroxy diphenyl ether
(triclosan) and benzoic acid; anti-inflammatory agents such as
acetylsalicylic acid and glycyrrhetinic acid; anti-seborrhoeic
agents such as retinoic acid; vasodilators such as nicotinic acid;
inhibitors of melanogenesis such as kojic acid; and mixtures
thereof. Further additional active agents including, for example,
alpha hydroxyacids, alpha ketoacids, polymeric hydroxyacids,
moisturizers, collagen, marine extract, and antioxidants such as
ascorbic acid (vitamin C), .alpha.-tocopherol (Vitamin E),
.beta.-tocopherol, .gamma.-tocopherol, .delta.-tocopherol,
.epsilon.-tocopherol, .zeta..sub.1-tocopherol,
.zeta..sub.2-tocopherol, .eta.-tocopherol, and retinol (vitamin A),
and/or pharmaceutically acceptable salts, esters, amides, or other
derivatives thereof. A preferred tocopherol compound is
.alpha.-tocopherol. Additional agents include those that are
capable of improving oxygen supply in skin tissue, as described,
for example, in Gross, et al, WO 94/00098 and Gross, et al, WO
94/00109, both assigned to Lancaster Group AG (incorporated herein
by reference). Sunscreens may also be included.
[0077] Other embodiments may include a variety of non-carcinogenic,
non-irritating healing materials that facilitate treatment with the
formulations of the invention. Such healing materials may include
nutrients, minerals, vitamins, electrolytes, enzymes, herbs, plant
extracts, glandular or animal extracts, or safe therapeutic agents
that may be added to the formulation to facilitate the healing of
dermal disorders.
[0078] The amounts of these various additives are those
conventionally used in the cosmetics field, and range, for example,
from about 0.01% to about 20% of the total weight of the topical
formulation.
[0079] The formulations of the invention may also include
conventional additives such as opacifiers, fragrance, colorant,
stabilizers, surfactants, and the like. In certain embodiments,
other agents may also be added, such as antimicrobial agents, to
prevent spoilage upon storage, i.e., to inhibit growth of microbes
such as yeasts and molds. Suitable antimicrobial agents are
typically selected from the group consisting of the methyl and
propyl esters of p-hydroxybenzoic acid (i.e., methyl and propyl
paraben), sodium benzoate, sorbic acid, imidurea, and combinations
thereof.
[0080] The formulations may also contain irritation-mitigating
additives to minimize or eliminate the possibility of skin
irritation or skin damage resulting from the chemical entity to be
administered, or other components of the composition. Suitable
irritation-mitigating additives include, for example:
.alpha.-tocopherol; monoamine oxidase inhibitors, particularly
phenyl alcohols such as 2-phenyl-1-ethanol; glycerin; salicylates;
ascorbates; ionophores such as monensin; amphiphilic amines;
ammonium chloride; N-acetylcysteine; capsaicin; and chloroquine.
The irritation-mitigating additive, if present, may be incorporated
into the compositions at a concentration effective to mitigate
irritation or skin damage, typically representing not more than
about 20 wt. %, more typically not more than about 5 wt. %, of the
formulation.
[0081] Further suitable pharmacologically active agents that may be
incorporated into the present formulations in certain embodiments
and thus topically applied along with the active agent include, but
are not limited to, the following: agents that improve or eradicate
pigmented or non-pigmented age spots, keratoses, and wrinkles;
antimicrobial agents; antibacterial agents; antipruritic and
antixerotic agents; anti inflammatory agents; local anesthetics and
analgesics; corticosteroids; retinoids; vitamins; hormones; and
antimetabolites.
[0082] Some examples of topical pharmacologically active agents
include acyclovir, amphotericins, chlorhexidine, clotrimazole,
ketoconazole, econazole, miconazole, metronidazole, minocycline,
nystatin, neomycin, kanamycin, phenytoin, para-amino benzoic acid
esters, octyl methoxycinnamate, octyl salicylate, oxybenzone,
dioxybenzone, tocopherol, tocopheryl acetate, selenium sulfide,
zinc pyrithione, diphenhydramine, pramoxine, lidocaine, procaine,
erythromycin, tetracycline, clindamycin, crotamiton, hydroquinone
and its monomethyl and benzyl ethers, naproxen, ibuprofen,
cromolyn, retinol, retinyl palmitate, retinyl acetate, coal tar,
griseofulvin, estradiol, hydrocortisone, hydrocortisone 21-acetate,
hydrocortisone 17-valerate, hydrocortisone 17-butyrate,
progesterone, betamethasone valerate, betamethasone dipropionate,
triamcinolone acetonide, fluocinonide, clobetasol propionate,
minoxidil, dipyridamole, diphenylhydantoin, benzoyl peroxide, and
5-fluorouracil,
[0083] A cream, lotion, gel, ointment, paste or the like may be
spread on the affected surface and gently rubbed in. A solution may
be applied in the same way, but more typically will be applied with
a dropper, swab, or the like, and carefully applied to the affected
areas.
[0084] The application regimen will depend on a number of factors
that may readily be determined, such as the severity of the
condition and its responsiveness to initial treatment, but will
normally involve one or more applications per day on an ongoing
basis. One of ordinary skill may readily determine the optimum
amount of the formulation to be administered, administration
methodologies and repetition rates. In general, it is contemplated
that the formulations of the invention will be applied in the range
of once or twice weekly up to once or twice daily.
BRIEF DESCRIPTION OF THE FIGURES
[0085] The invention will be further described in the following
examples with reference to the figures in which:
[0086] FIG. 1 shows the increase in transepithelial electrical
resistance (TEER) in keratinocytes during the differentiation of
keratinocyte. (A). Expression of claudin-4 (CLDN4) (A), occludin
(OCLN) (B) and zonula occludens-1 (ZO-1) (C) during the
differentiation of keratinocytes. Mean.+-.SE shown, *** p<0.001,
compared to 1-day sample).
[0087] FIG. 2 shows regulation of transepithelial electrical
resistance (TEER) in keratinocytes treated with 0, 10, 50, 100, 250
and 500 .mu.M of betaine for 1 h, 12 h and 24 h (A). Expression of
claudin (CLDN4) (B), occludin (OCLN) (C) and zonula occludens-1
(ZO-1) (D) in keratinocytes after 24 h treatment with 0, 10, 50,
100, 250 and 500 .mu.M of betaine. Mean.+-.SE shown, * p<0.05,
compared to sample with no betaine.
[0088] FIG. 3 shows regulation of transepithelial electrical
resistance (TEER) in keratinocytes treated with medium (CTRL), 10%
(vol/vol) de Man, Rogosa and Sharpe (MRS) broth used to cultivate
the probiotics, and 10% (vol/vol) soluble probiotic metabolites for
1 h, 8 h and 24 h (A). Expression of claudin (CLDN4) (B), occludin
(OCLN) (C) and zonula occludens-1 (ZO-1) (D) in keratinocytes after
24 h treatment with medium (CTRL), 10% (vol/vol) de Man, Rogosa and
Sharpe (MRS) broth used to cultivate the probiotics, and 10%
(vol/vol) soluble probiotic metabolites for 24 h. Mean.+-.SE shown,
*p<0.05; **p<0.01; ***p<0.001.
[0089] FIG. 4 shows regulation of transepithelial electrical
resistance (TEER) in keratinocytes treated with medium (CTRL),
probiotic whole cells or probiotic cell lysates for 1 h, 8 h and 24
h (A). Expression of claudin (CLDN4) (B), occludin (OCLN) (C) and
zonula occludens-1 (ZO-1) (D) in keratinocytes after 24 h treatment
with medium (CTRL), medium (CTRL), probiotic whole cells or
probiotic cell lysates for 24 h. Mean.+-.SE shown, *p<0.05;
**p<0.01, *** p<0.001.
EXAMPLES
[0090] In this study we differentiated normal human epidermal
keratinocytes in cell culture inserts, and investigated the effect
of betaine and probiotics, either soluble metabolites, whole
bacterial cells or bacterial cell lysates on barrier
characteristics. Transepithelial electrical resistance (TEER) is
widely used in epithelial cell studies to measure the tight
junction integrity. TEER reflects the transepithelial permeability
of water-soluble ions, and a higher TEER indicates a lower ionic
permeability. Moreover, we studied the expression of the claudin-4,
occludin and zonula occludens-1 in these cells.
Material and Methods
Keratinocyte Differentiation
[0091] Adult normal human primary keratinocytes (Invitrogen,
Carlsbad, Calif., USA) were maintained in EpiLife.RTM. medium with
60 .mu.M calcium (Invitrogen) supplemented with Human Keratinocyte
Growth Supplement (HKGS) (Invitrogen) (basal medium) at 5% CO.sub.2
atmosphere. To differentiate the keratinocytes, the P5-P7 cells
were plated on ThinCert.TM. cell culture inserts (Greiner Bio-one,
Frickenhausen, Germany) at density of 10.sup.5cells/cm.sup.2. After
overnight incubation at 5% CO.sub.2 atmosphere the basal medium was
aspirated and changed to differentiation medium (DM) consisting of
EpiLife.RTM. medium with 1.45 mM CaCl.sub.2 supplemented with human
keratinocyte growth supplement and incubated for 4 days at 5%
CO.sub.2 atmosphere. DM was changed both at both the apical and
basolateral side every day. Test substances were given at fourth
day after starting the differentiation.
Experiments With Differentiated Keratinocytes
[0092] Betaine (Danisco) were dissolved in DM, and applied to
apical side of differentiated keratinocytes at amounts of 0, 10,
50, 100, 250 and 500 .mu.M. The cells were incubated for 1, 12, and
24 hours at which points TEER was measured as described below.
[0093] Lactobacillus acidophilus NCFM.RTM. (ATCC 700396),
Bifidobacterium lactis 420 DSM22089 (commercially available from
Danisco A/S), L. acidophilus La-14 (ATCCSD5212) (commercially
available from Danisco A/S), L. salivarius Ls-33 (ATCCSD5208) and
Propionibacterium jensenii P63 (DSM 22192) (Danisco Cultures,
Paris, France) were cultured anaerobically at 37.degree. C. in de
Man, Rogosa and Sharpe (MRS) broth (LabM, Bury, United Kingdom).
For experiments with soluble probiotic metabolites the bacteria
were cultivated to OD600.apprxeq.0.6-0.9, and for experiments with
whole bacterial cells the cultivation continued until the
exponential growth rate of bacteria was achieved. The bacterial
cell densities were determined with flow cytometry (FACSCalibur,
Becton Dickinson, San Jose, Calif., USA). Bacterial cells were
removed from the soluble metabolites by centrifuging 25.degree. C.,
5 min, 3000.times.g. Bacterial pellets were discarded, and
supernatant containing soluble metabolites was diluted to 10%
(vol/vol) in DM, sterile-filtered with 0.2 .mu.m sterile syringe
filter units (Sartorius, Goettingen, Germany). Bacterial cells were
washed once with DM, and administered as 100 bacterial cells
towards one keratinocyte suspended in DM.
[0094] For studies with bacterial cell lysates the bacterial cells
were sheared with Precellys 24 bead beater (Bertin Technologies,
Saint-Quentin-en-Yvelines Cedex, France) with grinding kit VK01
(Bertin Technologies) at 6500 rpm with 3 cycles of 45 seconds. The
breakdown of the cells was confirmed by light microscopy. The
bacterial cell lysates suspended in DM were sterile-filtered with
0.2 .mu.m syringe units (Sartorius) before applying them to the
apical side of the differentiated keratinocytes at an amount that
corresponded to 100 lysed bacterial cells to one keratinocyte.
[0095] The differentiated keratinocytes were treated with soluble
metabolites produced by probiotics, probiotic whole bacterial
cells, and bacterial cell lysates from the apical side for 1 h, 8 h
and 24 h at which the TEER was measured. As a control for
experiments with soluble metabolites 10% bacterial culture media
was used. At the end of each experiment at 24 h time point the
keratinocytes were lysed for RNA isolation (see below). In some of
the experiments baseline TEER decreased towards the 24 h treatment
period due to probably variation in the cell amount plated in the
insert.
Measurement of TEER
[0096] The integrity of the monolayer was verified by measuring
TEER before and after each time point using Millicell-ERS system
(Millipore, Billerica, Mass., US) (26). The TEER obtained from the
monolayer and the insert was subtracted with background TEER
obtained from the insert to yield the monolayer resistance and
multiplied with the area of the insert to obtain the result as
ohm.times.cm.sup.2. For the experiments with betaine and probiotics
the results are expressed as a percentage change in TEER (% Change
in TEER) which reflects the change in the resistance in the given
time point compared to a control time point, which was the same
cell monolayer before applying the test substances.
RNA Isolation and cDNA Synthesis
[0097] The medium was aspirated after day 1, 2, 3 and 4 along the
keratinocyte differentiation process, or at the end of the
experiment after 24 hours for the experiments with betaine or
probiotics, and the cells were lysed immediately with buffer RA1
supplemented with 1% .beta.-mercaptoethanol (Sigma-Aldrich, St.
Louis, Mo., USA). Total RNA was isolated using NucleoSpin.RTM. 96
RNA kit (Macherey Nagel GmbH & Co. KG, Duren, Germany).
Reverse-transcription was done using SuperScript III (Invitrogen,
Carlsbad, Calif., US) with random primers (Invitrogen).
Real-Time Quantitative PCR
[0098] Real-time quantitative PCR of claudin-4 (CLDN4), occludin
(OCLN), zonula occludens-1 (ZO-1) was done with TaqMan gene
expression assays (Applied biosystems, Foster City, Calif., USA)
designed for each specific gene in an amplification reaction of 20
.mu.l according to manufacturer's instructions with 7500 Fast
Real-time PCR System (Applied Biosystems). As an endogenous control
gene ribosomal protein, large, P0 (RPLP0) was quantified in
parallel in each experiment (27, 28). The expression of RPLP0 was
first tested with extensive set of differentially treated samples,
and its expression was found to be constant in various treatments
(data not shown). The data was analyzed by using the
2.sup.-.DELTA..DELTA.Ct method (29), and the results are expressed
as fold increase of mRNA amount of the unknown samples relative to
the mRNA amount obtained for the sample at day 1 for keratinocyte
differentiation, or for untreated control samples for experiments
with betaine and probiotics.
Statistical Analyses
[0099] The statistical significance of differences between
treatments were determined using one-way ANOVA. P-values of 0.05 or
less were considered as significant. All comparisons were made
against the respective medium-only controls unless otherwise
stated. Correlation of the TEER and the expression of tight
junction components during keratinocyte differentiation was tested
with Pearson correlation calculation. All analyses were calculated
using GraphPad Prism version 5.01. Mean.+-.SE values are shown for
all the results.
Results
[0100] Differentiation of keratinocytes increased tight junction
integrity and expression of claudin-4
[0101] Normal primary human keratinocytes isolated from adults were
differentiated on cell culture inserts in order to follow the tight
junction formation between the epithelial cells by measuring
transepithelial electric resistance (TEER). Differentiation of
human adult primary keratinocytes in cell culture inserts increased
the TEER by 84%, 93% and 95% at day 2, 3, and 4 from the start of
the study (p<0.001 at all time points compared to day 1) (FIG.
1). The increase in TEER was time-dependent with Pearson r=0.7556
(p=0.0003).
[0102] During the 4-day differentiation process the gene expression
of tight junction components CLDN4, OCLN, and ZO-1 in the
keratinocytes was studied. The expression of OCLN and ZO-1 remained
at basal level throughout the differentiation process, whereas the
expression of CLDN4 increased (FIG. 1 b) (p<0.001 at day 4;
compared today 1). The TEER values correlated positively with CLDN4
expression (p<0.001, Pearson r=0.8093), whereas no correlation
between TEER and ZO-1 expression or TEER and OCLN expression was
observed.
Betaine increased keratinocyte tight junction integrity and
decreased the expression of occludin.
[0103] The effect of betaine on differentiated keratinocytes was
studied by incubating the cells for 1, 12, and 24 hours from the
apical side with 0, 10, 50, 100, 250 and 500 .mu.M of betaine. TEER
remained at the basal level with the lowest amount of betaine, but
increased after 1 hour with 50 .mu.M betaine (p<0.05) throughout
the higher concentrations, and remained increased still after 12 h
(p<0.05 to p<0.001) (FIG. 2 a). The baseline TEER increased
at 24 h, and only 100 .mu.M and 500 .mu.M amounts of betaine showed
an additional increase in TEER of 41.8.+-.2.8% and 47.8.+-.4.2%,
respectively (p<0.05 and p<0.01, respectively).
[0104] The expression of CLDN4 (FIG. 2 b), OCLN (FIG. 2 c) and ZO-1
(FIG. 2 d) in the cells incubated 24 h 0, 10, 50, 100, 250 and 500
.mu.M of betaine was determined. The expression of CLDN4, and ZO-1
remained similar to the basal levels in the betaine-treated
samples. Moreover, expression of OCLN decreased in samples treated
with 100, and 250 .mu.M betaine (p<0.05). In contrast to the
baseline differentiation in this data set, the amount of OCLN also
correlated negatively with TEER (p<0.05, Pearson r=-0.6214).
Probiotics increased keratinocyte tight junction integrity.
[0105] The differentiated keratinocytes were treated with soluble
probiotic metabolites, whole probiotic bacterial cells or probiotic
bacterial cell lysates for 1 h, 8 h, and 24 h.
[0106] Compared to 10% bacterial culture media control (MRS),
soluble metabolites samples increased the TEER values (FIG. 3 a).
At 1 h time point the TEER increased with all tested soluble
metabolites with an exception of P. jensenii P63 produced soluble
metabolites, which did not increase the TEER above the level of the
10% bacterial culture media control. The highest increase in TEER
with probiotic soluble metabolites was observed with the B. lactis
420 soluble metabolites, that increased TEER 20.4%.+-.2.8%
(p<0.001) at 8 h, and 80.3%.+-.3.8% (p<0.001) at 24 h.
[0107] The incubation of keratinocytes with whole bacterial cells
showed an increase in TEER (FIG. 4 a). TEER remained at the basal
level in keratinocytes treated by L. acidophilus NCFM.RTM., L.
salivarius Ls-33, and P. jensenii P63 at 1 h and at 8 h. At 24 h
the baseline TEER value of the control was noticeably decreased
from the value observed before the start of the experiment.
However, in L. acidophilus NCFM.RTM. (21.4%.+-.4.4%, P<0.01), L.
salivarius Ls-33 (42.2%.+-.24.4%, p<0.001), and P. jensenii P63
(33.1.+-.11.3%, p<0.01) the increased TEER above the baseline at
24 h was significant. In contrast, in keratinocytes treated with L.
acidophilus La-14 the TEER decreased below the baseline
significantly already at 1 h time point (-38.3%.+-.4.4%,
p<0.01), and remained decreased throughout the whole experiment
(-43.8.+-.4.7%, p<0.01 at 8 h, and -41.3%.+-.4.1%,
non-significant at 24 h).
[0108] In the keratinocytes treated with bacterial cell lysates
(FIG. 4 a) no similar decrease in TEER was observed in the samples
treated with L. acidophilus La-14, and B. lactis 420 lysates as was
observed in keratinocytes treated with respective whole cells.
However, compared to other probiotics these aforementioned as well
as P. jensenii P63 lysate had a smaller effect, as the TEER
increased significantly only after 24 h (15.2%.+-.3.3% for L.
acidophilus La-14 lysate p<0.05; 13.5%.+-.9.6% for B. lactis 420
lysate p<0.05; 25.7%.+-.8.0% for P. jensenii P63 lysate
p<0.01). L. acidophilus NCFM.RTM. lysate and L. salivarius Ls-33
lysate had a stronger effect since they induced an increase in TEER
already after 1 h (20.4%.+-.3.2% p<0.05, and 20.5%.+-.4.9%
p<0.05, respectively), that remained elevated at 8 h
(23.4%.+-.4.1%, p<0.05, and 18.3%+5.7% nonsignificant,
respectively), and at 24 h (43.6%.+-.3.0%, p<0.001, and
33.8%.+-.5.6%, p<0.01).
Probiotics affected the keratinocyte tight junction protein
expression strain-dependently
[0109] The expression of the TJ protein CLDN4, OCLN, and ZO-1 from
the cells treated 24 h with soluble metabolites produced by the
probiotics, whole probiotic bacteria as well as cell lysates was
determined.
[0110] In the case of soluble metabolites treated keratinocytes
gene expression changes beyond the changes in bacterial culture
media control was observed in the case of CLDN4 (FIG. 3 b) but not
with OCLN (FIG. 3 c) or ZO-1 (FIG. 3 d). The soluble metabolites
produced by three of the probiotics strains increased CLDN4
expression in soluble metabolites treated keratinocytes: L.
acidophilus La-14 (Fold difference (FD)=9.4.+-.0.3, p<0.05,
compared to bacterial culture media), L. salivarius Ls-33
(FD=12.4.+-.3.5, p<0.01, compared to bacterial culture media),
and P. jensenii P63 (FD=14.8.+-.1.3, p<0.01, compared to
bacterial culture media). B. lactis 420 soluble metabolites has
also tendency to increase CLDN4 expression (FD=8.6.+-.0.05), but it
did not reach significance. The expression of CLDN4 did not show
correlation to TEER values, but a weak correlation was observed
between ZO-1 and TEER (Pearson r=-0.5851, p<0.05).
[0111] In the case of whole bacterial cell treated keratinocytes,
no effect on regulation of CLDN4 (FIG. 4 b), OCLN (FIG. 4 c) or
ZO-1 (FIG. 4 d) was observed in these cells, as well as no
correlation to the TEER values.
[0112] However, when bacterial cell lysates were used to treat the
cells, L. acidophilus NCFM.RTM. lysate was observed to up-regulate
OCLN expression (FIG. 4 c) (FD=2.1.+-.0.3, p<0.001), and ZO-1
expression (FD=2.8.+-.0.2, p<0.01). From all the bacterial cell
lysates B. lactis 420 lysate was also able to increase the ZO-1
expression (FD=2.5.+-.0.3, p<0.05). No correlation between
CLDN4, and ZO-1 and TEER was observed in bacterial cell lysates
treated cells but a correlation between TEER and OCLN expression
was observed (Pearson r=0.7107, p<0.05).
Discussion
[0113] The tight junctions form a part of the epithelial
permeability barrier between the normal human epidermal
keratinocyte cells in culture. Permeation of solutes through the
intact TJ can be quantified by transepithelial electrical
resistance. In immunofluorescence studies, differentiation of
keratinocytes by a high Ca.sup.2+ concentration induced
translocation of occludin, claudin-1, and claudin-4 to overlapping
continuous lines circumscribing individual cells, indicating that
the proteins localized to TJs. We observed that the expression of
occludin, and ZO-1 were constant during the Ca2+ induced
differentiation of the epidermal keratinocytes, but the expression
of claudin-4 increased steadily. Previously it has been documented,
that when TJ structure and permeability barrier was disrupted with
ochratoxin A in normal human keratinocytes, the expression of
claudin-4 was reduced.
[0114] In a comparative study betaine was shown to increase the
TEER values but did not affect the expression of the tight junction
proteins studied here other than occludin. Removal of occludin by
siRNA technique resulted in decreased TEER (36), but knock-out of
occludin in mice do not alter the epidermal barrier function (37)
and it is quite possible that reduced expression of occludin is not
linked with increased TEER in betaine-treated keratinocytes even
though a weak negative correlation between occludin and TEER was
observed. An increase in TEER indicates that betaine improves tight
junction integrity, and could contribute to the inside-outside
barrier and hydration status of the skin also through this
mechanism additionally to its osmolyte function. The function of
organic osmolytes in regulation of barrier integrity is also
indicated by studies with taurine and betaine which reduced the
irritating effects of surfactants in epidermis (44).
[0115] Previous studies with probiotics have concentrated on either
systemic or gastrointestinal effects.
[0116] Soluble metabolites produced by probiotics were observed to
affect TEER and expression of tight junction proteins
differentially compared to whole probiotic cells or probiotic cell
lysates, a phenomenon that has also been reported for intestinal
epithelial cells. In this study, expression of claudin-4 was
regulated strongly by soluble metabolites produced by probiotics in
a strain-specific manner but not by whole probiotic cells or their
lysates.
[0117] In this study, up regulation of occludin was observed with
the probiotic L. acidophilus NCFM.RTM. cell lysate, in contrast to
down regulation with betaine. L. acidophilus NCFM.RTM. lysate was
able to regulate another TJ gene, ZO-1, which was also up regulated
also by B. lactis 420. ZO-1 and occludin have been observed
previously to be regulated by probiotics in intestinal epithelial
cells, and it has been shown that leaky gut was prevented in
dextran sodium sulfate--induced colitis in mice by a probiotic E.
coli Nissle 1917 through upregulation of ZO-1 expression (51). ZO-1
has been shown to stabilize the TJ barrier through a link between
the barrier and perijunctional actomyosin cytoskeleton (30). The
probiotic cell lysates increased the TEER values.
[0118] The topical application of probiotics would only affect the
uppermost skin layers, and thus, in order to achieve penetration
into the deeper layers, the cell lysates or soluble metabolites may
be better.
[0119] Tight junctions are important not only as barrier against
free diffusion of fluids, electrolytes and macromolecules, but also
against the translocation of surface associated micro-organisms and
their secreted products (Sousa et al 2005). Probiotic bacterial
cell lysates could be a used to enhance this barrier without
inducing pathological immune responses.
[0120] In conclusion we show that betaine, and probiotics are able
to improve TJ integrity between normal human epidermal
keratinocytes. Enhancement of the skin barrier may be important
particularly in diseases where barrier function is impaired.
[0121] It is to be understood that while the invention has been
described in conjunction with the preferred specific embodiments
thereof, the foregoing description is intended to illustrate and
not limit the scope of the invention.
[0122] Other aspects, advantages, and modifications will be
apparent to those skilled in the art to which the invention
pertains.
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