CA2106631A1

CA2106631A1 - Skin-care compositions

Info

Publication number: CA2106631A1
Application number: CA002106631A
Authority: CA
Inventors: Rolf-Dieter Petersen
Original assignee: Rolf-Dieter Petersen; Chemisches Laboratorium Dr. Kurt Richter Gmbh
Current assignee: Chemisches Laboratorium Dr Kurt Richter GmbH
Priority date: 1992-09-21
Filing date: 1993-09-21
Publication date: 1994-03-22
Also published as: EP0595012B1; DE4231544C1; ATE154234T1; EP0595012A1; ES2102334T1; DE59306731D1; ES2102334T3

Abstract

ABSTRACT

The invention relates to compositions for the protection of the skin, in particular for use in the prophylaxis of inflammations of the skin. The compositions of the invention contain cell wall fractions of gram positive Eubacteria.
Cell wall fractions of gram positive Eubacteria, whose base composition of the DNA contains a high guanine and cytosine portion (G + C mole % > 50%), such as those belonging to the bacterium species Micrococcus luteus, are preferred.

Description

2~ Q~3~

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.
'-.

, ~
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 :' : , ~: : . .:,;.
:
.:
: , .:

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.
.... ..
;,, 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 . .

2~LQ~3~

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.

:' ' .

: . ,~ , , : : : :. : :; . : : . :,: :

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.
. .
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.
,;': .' Different biological activities of cell wall components of different microorganisms have already been described.
, 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.
~., 21Q~31 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.
.
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 .

'. .:

2~Q6631 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_.

2~0~631 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.
,: ' ~Mammal skin is considered to be the "primary natural habitat" of Micrococcus luteus.
.
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.
.
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, ,,",,.

2~L~6~

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.~
.
~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 , ~. .

. . ~ . ,. : ~. . . : , : .

2 ~ 3 ~

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.
~: ~
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:
. .
:
" .

,. , . , ., :, .. .. ,,, . , ,. .. , . . ~ : . ., . . ,. ~ . ,: . . : :.::

-` 21~fi31 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:
, 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.
:':
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) , ~

21Q~631 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:
, 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.

;, , .
, " .' :; " :

21Q6~3~ :
12 .

... .

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 % :
.
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 %

: : : : .
~: :, .
:: ' ~ -.,,.:
:

2~0~6~1 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 %
" ' paraffinum subliquidum 5.0 %

etearyl alcohol 5.0 %

a preservative q.s.

~b) water, distilled 44.4 %
. :' : a preservative~ q.s.

glycerin : ~ 5.0 %
, ~ sodium chloride; : 0.5 % .~ .
: ; : ..: , ~ c)~ cell wall fractions ~ 10.0 %

i , .:
-: :

: :

21Q~63~ ~

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.

~ .:

' ' . . , .. , . ........ , .. .. .. .,, . ~ . ; - - ~ ,. . ..

: ' . ; : . . ' ;, ' ' ' , ;, . . : ,. ':' , ' .' ' ' ' '

Claims

1. A skin-care composition comprising cell wall fractions from gram positive Eubacteria, the cell wall fraction being obtainable by disrupting the cells, separating the cytoplasm and subjecting the cell wall to enzymatic hydrolysis.

2. The composition according to claim 1, wherein the DNA
base composition of the gram positive Eubacteria has a guanine and cytosine portion (G + C mole %) which is greater than 50%.

3. The composition according to claim 1, wherein the gram positive Eubacteria is non-pathogenic.

4. The composition according to claim 1, wherein the gram positive Eubacteria belongs to the species Micrococcus luteus, Cellulomonas flavigena, or Arthrobacter globiformis.

5. The composition according to claim 1, wherein the enzymatic hydrolysis of the cell wall is carried out using trypsin and lysozyme.

6. A skin-care composition containing cell wall fractions from Micrococcus luteus, Cellulomonas flavigena, or Arthrobacter globiformis.

7. The composition according to claim 6, comprising a prophylactically effective amount of the cell wall fractions in a topically acceptable carrier.

8. A method for protecting the skin from accelerated ageing effects of UV light or irritants comprising applying to the skin a composition comprising a prophylactically effective amount of cell wall fractions from gram positive Eubacteria.

9. The method according to claim 8, wherein the DNA base composition of the gram positive Eubacteria has a guanine and cytosine portion (G + C mole %) which is greater than 50%.

10. The method according to claim 8, wherein the gram positive Eubacteria is non-pathogenic.

11. A method according to claim 8, wherein the gram positive Eubacteria belongs to the species Micrococcus luteus, Cellulomonas flaviqena, or Arthrobacter globiformis.