Note: Descriptions are shown in the official language in which they were submitted.
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The invention relates to skin-care compositions for the
protection of the skin, in particular for use in the
prophylaxis of inflammations of the skin. The compositions
according to the invention contain cell wall fractions of
gram positive Eubacteria. Gram positive Eubacteria, whose
base composition of the DNA shows a high guanine and
cytosine portion (G+C mole % > 50%), in particular those of
the bacterium species Micrococcus luteus, are preferred.
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It~ has been known for quite some time that UV absorbers andspeciic antioxidants which are useful in the prophylaxis of
skin lesions and accelerated light-induced skin ageing play
an important role in cosmetic~s as light protective agents.
Recent a~dvances in~ photochemistry~ which shed light on
nflammatory~processes occurring ~on~ a cellular level show
;that~the topical application of particular substances can ~e
effectively used in~the prophylaxis of light-induced skin
agelng. Epldermis cells react to UV rays in many wavs, which
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is referred to as the "inflammatory response". A topical or
systemic inflammatory reaction upon exposure to W B
radiation has been described. This reaction was prompted by
the release of cytokines, such as interleukin 1 and 6 ~IL-1,
IL-6), tumor necrosis ~actor ~ (TNF-~) and other mediators.
A significant decrease in the release of IL-6 after
application of corticosteriods as anti-inflammatory agents
has been observed.
Moreover, inflammatory reactions can be prompted not only by
W B but also by WA radiation and PW A treatment (psoralen
plus W A treatment). Free radicals lead to the peroxidation
o~ cell membrane lipids. The activation of phospholipase
results in the release of arachidonic acid which in turn
metabolizes via cyclooxygenase into prostaglandines,
thromboxanes and prostacyclin on the one hand and via
lipoxygenase into leukotrienes and different mono-, di- and
trihydroxylated eicosatetraenoic acids (HETE's) on the other
hand.
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The inflammatory reaction induced by the release of
arachidonic acid simultaneously reduces the activity of the
antioxidative enzymes catalase and superoxide dismutase
(SOD). This occurs not only after W B radiation, but also
after W A radiationO
Components of skin-care or pharmaceutical compositions
which possess irritation or sensibilization potentials that
can prompt inflammatory reactions are also known to be
factors contributing to accelerated skin ageing.
Apart from this, W rays can directly ca~alyze reactions
even when these reactions have not been prompted by
inflammatory processes. The thymine structural units of a
strand which are adjacent to each other due to the helical
con~ormation of the DNA dimerize when exposed to light. The
endogenous enzymatic repair system of the cell is able to
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detect this damage, remove the segment with the dimer and
replace it correctly after a de novo synthesis ~excision
repair). If this repair process is reduced due to age or is
strained too much by excessive radiation, then this thymine
dimer, because of a distortion of the spatial arrangement of
the DNA, can cause mutations. Skin-care compositions
containing inactivated cultures of Bifidobacteria promoting
the natural DNA repair process are described in EP-A-43128.
Among the many possibilities of light-induced damage
accompanied by accelerated skin ageing, the inflammatory
reactions occurring on a cellular level are of special
importance. Inflammations prompted by W radiation,
irritants or toxic agents liberate different mediators, such
as histamine, prostaglandines or leukotrienes. They are
accompanied by epidermal cell injuries (sunburn cells), the
formation of apoptotic cells, and DNA damage, leading to the
formation of malignant melanomas or basal cell carainomas.
Additionally, degenerative processes occur, such as actinic
degeneration: Elastic fibers transform to lumpy amorphous
materials; collagen fibrils lose elasticity due to
crosslinkages.
Irritations continuing over a prolonged time which lead to
chronic inflammations are particularly critical. A forced
new skin formation i5 achieved by increased cell division.
HOWQVer, since the repair system is halted to a yreat extent
when the complicated cell division process takes place, the
DNA will be damaged and consequentIy the genetic material
will undergo mutations when exposed to irritants over a
prolonged time.
This relationship shows the importance of substances that
are useful in the prophylaxis of inflammations o~ the skin.
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Inflammatory processes of the skin can for instance be
treated by topically applied corticosteroid or acetyl
salicylic acid preparations. Since these substances, upon
resorption, are distributed in the whole body via the blood
circulation, they are said to have a systemic ef~ect.
Substances with a systemic effect are barred from use in
cosmetics, as they may have numerous undesirable side
effects.
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Fractions of plant extracts possessing anti-inflammatory
activity are known.
Substances which participate in the formation of cell walls
have been found to be particularly active.
While the cell walls of plants mainly consist of
polysaccharides and lignine, the structure of the c~ll wall
of for instance gram positive bacteria is considerably more
differentiated. The cell walls of gram positive bacteria
contain, apart from polysaccharides, structurally greatly
varied compounds, such as the peptidoglycans ~mureines) and
"accessory polymers", such as the teichoic acids, teich-
uronic acids or other amphiphilic macromolecules.
.
Consequently, the great variety of biological cell wall
components of gram positive bacteria provides a greater
potential for obtaining fractions with specific properties.
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Different biological activities of cell wall components of
different microorganisms have already been described.
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EP 0 201 332 for instance, which is fully incorporated
herein by reference, describes an anti-infectious effect in
intestinal infections achieved with polysaccharides made
from cell walls of Bifidus bacteria.
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These biologically active fractions of microorganisms
concern the pharmaceutical field, given their medical use.
The present invention pursues the object of providing skin-
care compositions for the protection of the skin, in
particular for use in the prophylaxis of inflammations of
the skin, which are able to make an important contribution
to the protection of the skin from accelerated ageing due to
the action of W light or irritants.
It has surprisingly been found that cell wall fractions of
gram positive Eubacteria possess a highly prophylactic
effect against inflammations of the skin. Gram positive
Eubacteria whose base composition of the DNA has a high
guanine and cytosine portion tG+C mole % > 50%), such as
those of the bacterium species Micrococcus luteus, are
preferred.
Consequently, the invention relates to the topical use of
cell wall fractions from gram positive Eubacteria. The
compositions may be applied topically to humans and animals.
They may be administered for example in the form of a creme
or lotion.
The invention also relates to skin-care compositions
containing cell wall fractions of gram positive Eubacteria,
the cell wall fraction being obtainable by cell disruption,
separation of the cytoplasm and enzymatic hydrolysis of the
cell walls.
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The isolation of cell wall fractions from gram positive
bacteria is advantageous over that from plants because of
the superior reproducibility of the starting material
because of the use of biotechnological processes. Moreover,
in the case of bacteria, the separation of particular cell
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wall fractions is easier from a manufacturing point of view,
because in the case of plant extracts the number of
undesired accompanying substances that have to be removed is
substantially greater.
Moreover, plants are subject to factors such as the degree
of ripeness, soil conditions or climate which influence
their quality.
Fig. 1 of the enclosed drawing shows the gel permeation
chromatogram of the cell wall lyophilisate of Micrococcus
luteus.
The basic division of gram positive Eubacteria (not to be
confused with the genus Eubacterium, which however also
belongs to the gram positive Eubacteria) into high and low-
GC-subdivisions has only recently been established and was
phylogenetically defined by Woese et al.
(Woese, C.R., Stackebrandt, E., Macke, T.J., and Fox, G.E.
1985. "A phylogenetic definition of the major eubacterial
taxa". System. Appl. Microbiol. 6:143-151).
The genus Micrococcus, and of this the species Micrococcus
luteus, has been found to be particularly suitable.
Bergey's Manual of Systematic Bacteriology, vol. 2, 1986,
pp. 1003-1008, describes, inter alia, the nutrient
requirements and the taxonomic characteristics of
Micrococcus luteu_.
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The cell wall of Micrococcus luteus is composed mainly o~
thick, rigid peptidoglycan layers. Schleifer and Kandler
found altogether six different types of peptidoglycans in
the cell walls of Microccoci.
(Schleifer, K.H. and 0. Kandler, 1972. "Peptidoglycan types
of bacterial cell walls and their taxonomic implications".
Bacteriol. Rev. 36; 407-477).
Teichoic acid was found to be absent, while teichuronic acid
was found to be present. (M. Yamada, A. Hirose and M.
Matsuhashi, 1975. "Association of lac~ cell wall teichuronic
acid with formation of cell packets of Micrococcus
lysodeikticus (luteus) mutants", J. Bacteriol. 123: 678- ~ -
686).
Each of the documents cited above is hereby fully
incorporated herein by reference.
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~Mammal skin is considered to be the "primary natural
habitat" of Micrococcus luteus.
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The active ingredient in the skin--care composition can be
prepared in the following manner, for instance from the
species Micrococcus luteus:
As an aerobe, Micrococcus luteus is cultured under usual -
growth conditions and is pasteurized for 30 to 60 minutes at
60-65C upon reaching the early stationary growth phase.
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The cells are harvested by centrifuging (5 minutes, at
5000 x g). A sterile physiological salt solution or
preferably a sterile, neutral 0.1 molar phosphate buffer is
used for washing which is carried out two or tree times.
Cell disruption is carried out using conventional mechanical
procesues, for instance ultrasonic waves, a cell bead mill,
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a high pressure homogenizer or a combination of said
processes.
The success of the disruption can be observed in a phase
contrast microscope providing a 1000-fold enlargement.
The cytoplasm is separated from the cell wall by
centrifugation (20,000 x g for 20 minutes). The sediment is
washed twice with 0.01 of molar sterile phosphate bu~fer, at
p~ 7Ø ~he cell wall material is lyophylized.
The isolation of active oligopeptidoglycans (mureine
components) and polysaccharides is carried out by
enzyma~ically lysing the crude cell wall material impurified
by proteins and nucleic acids.
The first step of the enzy,matic cell wall hydrolysis is
carried out using proteases, for instance trypsine, in order
to degrade cytplasmic proteins and membrane proteins.
The lyophilized crude cell walls are suspended in 50 mM
tris-HCl-buffer (pH 7.6), using ultrasonic waves for a short
time. A~ter the addition of trypsine, degradation occurs
under conventional conditions. Subsequently, the material is
subjected to centrifugation for about 20 minutes at 20,000
x g and the sediment is libera~ed from trypsin by repeated
washing with the afore-mentioned ~uffer solution. The gel-
like ~consistency of the sediment is attributable to the
presence~of nucleotides which can ~e degraded by nucleases
using conventional methods.~
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~A~ter the elimination of impurities, the cell walls are
solubilized with the aid of lysozyme. The cell wall fraction -,
is~ suspended in ~50 mM tris-~Cl-buffer (pH 7.6) using
ultrasonic waves and lysozyme is added in the required ,-
amount. The hydrolysis Or the cell wall is finished when the
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transmission measurements of the samples no longer show an
increase.
The lysozyme is inactivated preferably using proteinase K,
which is in turn inactivated by being heated to about 80~C
for a short time.
After dialysis and lyophilization, the cell wall fractions
are present in the desired soluble form.
The analytical characterization of the cell wall
lyophilisate is carried out using gel permeation
chromatography (see Fig. 1). ~ -
Column: Nucleogel\GFC 300-8 Macherey Nagel
Element: HPLC-water containing 0.02% of NaN3
Flow: 1 ml/min.
Detector: R I
Polymer-standard: Pullulan
((Dalton) 5,800-186,000)
Result: The soluble cell wall is composed of
fractions having an average molecular
weight distribution between 10,000 and
200,000 Dalton. (Exclusion limit of the
separation column at approximately
300,000 Dalton3.
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Proof of effectiveness:
In-vitro tests in cel~l cultures -
The tests were carried out with cell wall fractions of the
foIlowing gram positive Eubacteria, whose base composition
of the DNA is characterized by a high portion of guanine and
cytosine (G+C mole ~ > 50%), the fractions having been
prepared according to the above-described methods:
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Cellulomonas flaviqena
Arthrobacter alobiformis
Micrococcus luteus
The objective of the tests can be put into practice in
macrophage cultures, as macrophages play a fundamental role
in inflammatory processes.
Macrophages react to many exogenous stimuli by releasing
pro-inflammatory substances (endogenous mediators) which
are: cytokines, prostaglandines, leukotrienes, hydroxy-
linolic acids and reactive oxygen species.
Test schedule:
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Time~ "~ ding: - First, the dose/effect
relationship and the time kinetics for the induction of the
cytokines tumor necrosis factor ~ (TNF), and interleukin 6
(IL 6) and the lipid mediator prostacyclin (PGI2) we~e
ascertained in cell cultures of tha murine macrophage cell
lina (J 774~. The cultures were incubated with doses ranging
from 1 ~g to 1 mg of dry mass per ml (log dilutions) ~or 1,
2,~4 and 24 hrs and TNF (bioassay), IL 6 (bioassay) and PGI2
(RIA,~ as 6-keto-PGF2~) were determined in the supernatants.
The ~test was carried out in microtiter plates in
tripllcates, separately for the TNF/IL 6 assay and the PGI2
assay. ~ ~
Inh~ition o~ the formation of medi tors: - J 774 - cell
cultures were stimùlated with endotoxin (lipopolysaccharide,
LPSj~ in the presence of the cell wall fractions.
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Subsequently, the TNF content in the supernatant was
determined. The cell wall fractions,~again in concentrations
ranging from 1 ~g to 1 mg of dry~mass per ml (log dilutions)
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were pre-incubated for 0, 1, 2, 4 and 24 hrs, stimulated
with LPS, and TNF/IL 6 was determined in the supernatants.
The test was carried out in microtiter plates in
triplicates. Again, separate test were run for the effect on
the synthesis of PGI2.
Suprisingly, the cell wall fractions prepared from
Micrococcus luteus were found to possess the ~est anti-
inflammatory effect.
A test was simultaneously run in macrophages to find out
whether the active cell wall fractions themselves induce
inflammatory reactions. No release of cytokines after
exposure to cell wall fractions from Micrococcus luteus was
found, thus showing that the active ingredient of the
invention does not induce any pro-inflammatory reactions.
Examples of the skin-care compositions:
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The active ingredient of the invent:Lon - cell wall fractions
~rom Micrococcus lu_eus, dissolved in a weakly acidic buffer
solution, can be incorporated into many different topical
dosage forms in combination with the usual topical use
components and base materials. The compositions typically
co~prise an effective amount of the cell wall fractions in a
topically acceptable carrier, such as a lotion or creme.
They may be formulated in the form of a cosmetic.
The following formulations of the skin-care compositions
were chosen as examples. These examples are for purposes of
illustration only, and are not to be construed as limiting
the ecope of th~ invention in any way.
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Example 1
Cream O/W ':
a) a mixture of partial glycerides, fatty
alcohols, wax esters and ethoxylated
fatty alcohols (EmulgadeR SE) 3 %
capryl-capric acid esters of saturated
fatty alcohols (CetiolR LC) 5
partial glycerides and esters of long
: chain fatty acids (CutinaR BW) 2 %
stearyl alcohol 3 % :
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a preservative q.s.
b) water, distilled 71.9 %
:: a preservative ~ g.sO
glycerin 3.0 %
polyaorylate:(CarbopolR 954) 0-3 %
potassium hydroxide (10%) ~ 1.2 %
c)~: cell wall fractions ~ ~ lO.O %
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Example 2
Cream W/O
a) polysiloxane-polyalkyl-polyether-
copolymer (AbilR EM 90) 2.5 %
oleyl-erucate (CetiolR I 600) 8.0 %
isoproplymyristate 14.0 %
beeswax 5.0 %
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paraffinum subliquidum 5.0 %
etearyl alcohol 5.0 %
a preservative q.s.
~b) water, distilled 44.4 %
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: a preservative~ q.s.
glycerin : ~ 5.0 %
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~ sodium chloride; : 0.5 % .~ .
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~ c)~ cell wall fractions ~ 10.0 %
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Example 3
Gel
a) glycerin polyacrylate (HispagelR 200) 20.0 %
xanthan gum (1%) 30.0 %
water, distilled 35.7 ~
a preservative q.s.
oleyl-erucate (CetiolR I 609) 4.0 %
b) rell wall fractions 10.0 %
While a number of embodiments of this invention are
described herein, it is apparent that these embodiments may
be altered to provide other embodiments which utilize the
processes and products of this invention. Therefore, it will
be appreciated that the scope of this invention is to be
defined by the appended claims, rather than by th~ specific
em~odiments which have been presented by way of example.
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