AU619399B2 - Cosmetic composition containing compounds obtained by the culture of cells, especially fibroblasts or keratinocytes - Google Patents

Abstract

Cosmetic composition for treatment of the skin and the hair containing protective substances synthesised by cells cultured in vitro in response to at least one stress factor such as ultraviolet radiation, temperature variations, electromagnetic field, chemical agent or deficiency of an element normally present in the culture medium. The composition contains the culture medium containing or not containing the cells, the cells, or the solution obtained by bursting the cells. These compositions protect the skin and the hair in vivo against the stress factors undergone by the cells in vitro.

Description

COMMONWEALTH OF AUSTRALIA IFom1 PATENTS ACT 1952-69 COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Apnlinatlon Number: 53710/90 Lodged: 23.04.1990 Complete Specification Lodlged: Accepted: Published: **Priority *RFelated Art 0 oi, Name of Applicant:

L'OREAL

6 *AdJdress of Applicant Actual lnv,%ntor Address for Servie 14, rue Royale, 75008 Par~is, France ETIENNE SOUDAN!,. ELISABE7TI PICARD and ROLAND R(XUFT WATERMARK PAi ENT TRADEMARK ATTORNEYS.

LOCKED BAG NO. 6, HAWTHORN, VICTORIA 3122, AUSTRALIA Complete Specification for the Invention entitled: COSMETIC CCMPCSITION CONTAINING COMPOUNDS OBTAINED BY THE CULTURE OF CELLS, ESPECIALLY FIBROBLASTS OF KERATINmYTES The following statement Is a full descrlp';don of this Invention, Including the best methoc of performing It known to S-1 COSMETIC COMPOSITION CONTAINING COMPOUNDS OBTAINED BY THE CULTURE OF CELLS, ESPECIALLY FIBROBLASTS OR KERATINOCYTES This invention relates to a composition containing compounds obtained by cell culture.

A composition prepared by culturing cells in an appropriate I growth medium and suitable for cosmetic treatment is already disclosed in the document FR-A-2 585 567. The cells are preferably fibroblasts and keratinocytes. They can be human cels, despeciated animal cells, or even plant cells, and can be living or dead.

A cosmetic composition containing a growth medium obtained after isolation of the cells cultivated there is also known from EP-A-0 272 920.

These cosmetic compositions containing cultured cells together with their growth medi.il or containing solely the cell growth medium can transfer to the skin cellular vectors of a growth medium and, consequently, promote skin growth. However, they play no role in protecting the skin.

This invention relates to a cosmetic composition for protecting S" the skin or the hair against stresses of any kind, such as ultraviolet radiation, variations in temperature, dryness of the air and pollution.

According to this invention, protective substances synthesised by in vitro cultured cells subjected to stress during said in vitro culture are introduced into the cosmetic composition

I

2 intended to provide protection.

It is known that in all situations of stress encountered, e.g.

by the skin, the biological equilibrium must be maintained.

The skin therefore synthesises substances serving fcr its adaptation so as to maintain its protective role. However, these substances are not in themselves sufficient to protect the skin and in all cases the stress will have negative effects, owing, among other things, to the latent period elapsing between the attack and the synthesis of the protective substances by the skin. The same thing applies in the case of S the hair. By applying the cosmetic composition according to the invention, the skin and/or the hair will be in contact, at the time of the in vivo attack, with the protective substances *o prepared in vitro by cell culture. It has been established that substances synthesised in vitro can be utilised in vivo by the skin or the hair by external application. Moreover, it has been established that substances synthesised in vitro have a sufficient life span to be used in cosmetic compositions and, furthermore, that they are compatible with the different compounds generally used in cosmetic compositions.

Therefore, this invention relates to a cosmetic composition comprising, in a cosmetically acceptable -ehicle, at least one compound obtained by the culture of animal cells, especially fibroblasts or keratinocytes, characterised in that the said i compound is a substance protecting the skin or the hair, synthesised by the said cells in response to at least one situation of stress during in vitro culture.

The protective substances are preferably used without prior separation or purification, in the form of an active principle C composed of the culture medium and/or the cultured cells and/or the solution obtained after splitting of the cultured cells.

The culture medium and/or the solution obtained after splitting of the cells are advantageously concentrated to increase the protective substances content.

The composition can advantageously comprise a mixture of active principles obtained in response to several attacks. E.g. it is possible to introduce into the composition a mixture of the Sactive principles obtained by the culture of cells stressed by *S.o the action of ultraviolet rays and by the culture of cells Sstressed by heat shock.

S The attack preferably consists of the action of ultraviolet S rays, variations in temperature, an electromagnetic field, a chemical agent and/or a deficiency of an element normally present in the culture medium. According to the invention, the in vitro cells can be subjected to several attacks in succession. It is also possible to subject the cells to simultaneous attacks chemical attack and thermal attack).

In the case of multiple stress, the active pripciple obtained will directly comprise the substances obtained in response to 0 the different attacks, with no need for mixing.

The protective substances or mixtures of protective substances :@,Goo synthesised differ according to the stress applied to the cells. In general, these substances or mixtures of substances have not been Lsolated and are not yet well known. The protective substances are either excreted into the culture medium or localised in the cell. These protective substances can be molecules with a high energy content, substances having an immunological regulatory function, substances adapted to 4 promote cell growth and/or repair molecules.

It is known that cultures of enidermal cells subjected to the action of ultraviolet radi'tiJn synthesise, among other things, a hormone-like factor known as ETAF (Epidermal cell-derived Thymocyte Activating Factor), which can be assimilated to interleukin-1 (see articles in The Journal of Investigative Dermatology, Volume 81, No. 6, 1983; British Journal of Dermatology, 1985, 113, Supplement No. 28; and Archives of Dermatological Research, 1988, 280, pp. 71-76). Other cytokines are also produced by epidermal cells, in particular interleukin-3 and ENKAI (Epidermal cell-derived Natural Killer S cell Augmenting Factor).

S* Thermal attack results in the known manner in the synthesis of particular proteins, known as "heat shock" proteins, as a result of the fact that when the cells in culture are subjected to a sharp increase in temperature, a number of cellular proteins are denatured. The heat shock proteins then intervene to limit this denaturation and may break down abnormal proteins 0 and protect cellular structures, such as the cytoskeleton.

Electromagnetic stress gives rise to protective substances I which increase the permeability of membranes and stimulate certain metabolic pathways, such as the synthesis of adenosine S triphosphate.

Other different substances can be synthesised according to the stress applied to the cells.

According to this invention, the cells cultivated are preferably extracted from human skin, from the shaft of a human r hair or an eyelash, especially keratinocytes or fibroblasts.

The cells are advantageously taken from the person intended to use the cosmetic composition. It is possible in this manner to obtain personalised compositions, the application of which offers a very high degree of safety and innocuousness, and reduces the risk of allergy. It may be advantageous to take samples from the individual at an early age, then to freeze the cells after their multiplication and keep them for several years. After thawing, they would be placed in culture once I again, under the same conditions as recently extracted cells.

According to this invention, the cells are placed in culture and are then subjected to the selected stress (or stresses), preferably when they have reached confluence. The culture can be effected in the presence of precursors of the substances Sthat will be synthesised as a result of the stress. Examples of possible precursors are amino acids, nucleic bases and s sodium pyruvate.

A protocol of the culture of human keratinocytes and of the i culture of human fibroblasts will now be given by way of example.

a) Human keratinocytes culture protocol.

When keratinocytes derived from the epidermis are to be cultured, the skin is obtained, e.g. by means of conventional cosmetic surgery techniques. After being degreased, the epidermis is isolated by means of trypsinisation and the cells are placed in culture in a medium containing, e.g. 10 of foetal calf serum, antibiotics, pyruvate and glutamine.

I c L 6 When keratinocytes derived from the hair shaft are to be cultured, the hair follicles are collected in the anagLi phase by extraction or following cosmetic surgery. They are i cultured on a bovine crystalline membrane or on collagen in a modified Eagle's medium or a Mac Coy medium, supplemented with foetal calf serum (10 glutamine, antibiotics, fungicides, insulin, cholera toxin and EGF (Epidermal Growth Factor).

Keratinocytes can also be derived from the shaft of an eyelash.

b) Human fibroblasts culture protocol.

*0* Fibroblasts are isolated from the glabrous skin of the scalp.

After the hypodermis and the epidermis have been removed, the dermis is incubated in a solution of antibiotics and then washed in Phosphate Buffered Saline (PBS) and collagen-treated at 37 0 C. After the cellular suspension has been filtered and centrifuged, the residue is placed in culture in a complete culture medium at 2.104 cells/cm 2 The culture medium preferably consists of Dulbecco's modified S Eagle's medium supplemented with foetal calf serum (10 j penicillin, streptomycin, fungizone and glutamine.

jIt is possible to culture fibroblasts and keratinocytes simultaneously.

According to the protocols given hereinabove, the cells are then left to incubate in a drying oven maintained at a temperature of 37 0 C, in an atmosphere of 5 CO 2 and 95 relative humidity.

1 7 Different protocols applying to different stresses will now be Sgiven by way of example: 1) Stress by ultraviolet rays.

At confluence, the culture medium is eliminated, the cellular tapetum is washed with a buffered saline solution and then the cells are exposed to ultraviolet rays.

The doses of ultraviolet rays generally applied vary between 0.2 mJ/cm 2 and 75 kJ/cm 2 in particular, as follows: for keratinocytes: V 2 U.V.B. 1 to 100 mJ/cm 2 s 9* for fibroblasts: U.V.A. 2 to 75 kJ/cm 2 U.V.B. 5 to 30 mJ/cm 2 U.V.C. 0.2 to 0.5 mJ/cm 2 0 i After irradiation, a culture medium which may or may not contain foetal calf serum, growth factors and/or antibiotics is added to the cells and left in contact with said cells for a period that can vary from 12 to 48 hours, a period during which the cells respond to the stress by the synthesis of new molecules.

2) Stress by temperature.

It is easy to subject cultured cells to stress by temperature.

The following process is described by Nancy C. COLLIER and I II Milton J. SCHLESINGER in The Journal of Cell Biology, Vol. 103, October 86.

At confluence, the starting medium at a temperature of 37( is i replaced by another medium containing HEPES Shydroxyethylpiperazine-N-ethane sulphonic acid). The j ifibroblasts are placed in a 45°C thermostated water bath rith I stirring for a period varying from 30 minutes to three hcurs.

The medium is then replaced by modified Eagle's medium which may or may not contain foetal calf serum, and is then left in contact with the cells for 12 to 48 hours.

i, e The cells can be subjected to several heat shocks in S succession.

0* *e 3) Electromagnetic stress.

The culture medium is subjected to a homogeneous magnetic field of less than 20 gauss, ha-ring a peak with a duration of milliseconds and a frequency of less than 100 Hertz. The said magnetic field is obtained by employing a pair of Helmholtz coils surrounding the cell cultures at confluence. The culture medium is changed before passage into the electromagnetic S field. The application time of the field can range from 1 to 4 days. A process of this type is described by BERG in the i journal Studia Biophysica, Volume 119, 1987, Nos. 1-3.

4) Chemical attack.

Chemical attack can be achieved with the aid of detergents, heavy metals or salts.

-f i Stress by deficiency.

This is achieved by eliminating from the culture medium those elements normally present, such as amino acids, vitamins, sugars, nucleic bases, growth factors, foetal calf serum lipids and the serum itself.

When the cultures have been subjected to the desired stress or stresses, the protective substances produced in response to the said stress(es) are recovered- They are contained in the cells themselves (as in the case, e.g. of heat shock proteins) and/or in the culture medium (as in the case, e.g. of cytokines and growth factors).

*to If it is desired to recover the cells, the culture medium is Seliminated by aspiration. The cells are rinsed, then scraped from their vehicle and recovered in phosphate buffered saline.

They are then pulverised or lyophilised and introduced into cosmetic compositions. If it is desired to isolate the synthesised protective substances from the cells, the cells are split by various known techniques, such as sonication, osmotic shock, potting. The resulting suspension is then centrifuged and, as appropriate, the supernatant and/or the residue are recovered as an active principle to be incor.porated into the cosmetic compositions according to the invention.

If it is desired to recover the culture medium, centrifuging is effected after aspiration of the medium in order to eliminate the cells present. It is then possible to evaporate down the supernatant so as to potentiate the effects of the substances present.

According to the invention, the cosmetically acceptable vehicle of the composition can contain, in addition to the active principle(s) defined hereinabove, at least one cosmetically active compound which can be employed in the known manner as a constituent of a cosmetic composition. The cosmetic composition can be in the form of a cream, milk, lotion or serum. When preparing a cream, 0.1 to 30 by weight of the active principle(s) can be introduced into the cosmetic composition. In the case of a cosmetic composition in the form of a serum, the quantities of the active principle(s) introduced can be much higher, especially between 30 and 100 On average, 1 to 2 mg/cm 2 of the composition should be applied, generally morning and evening, this quantity being less (approximately 0.02 mg/cm 2 in the case of a serum or milk.

A clearer understanding of the invention will be obtained from the following examples, given purely by way of an illustration and being in no way limiting.

EXAMPLE 1 A process for the preparation of an active principle containing protective substances will now be described.

1. Culturing of keratinocytes In a first stage, keratinocytes derived from the mamilloplasty of a woman aged 40 years were cultured.

After being cut by a 0.3 mm electrokeratome, the epidermis was separated from the upper dermis by immersion in a trypsin I C~ r C- I I solution having a concentration of 0.20 The epidermis and Ithe trypsinisate were then harvested, subjected to stirring for 1 minute and tb ,i filtered through two layers of sterile gauze.

The keratinocytes were then seeded in a Dulbecco's modified Eagle's medium, containing 10 by weight of foetal calf serum, 1 by weight of non-essential amino acids, 1 by weight of sodium pyruvate and antibiotics (penicillin 75 U.I./ml, streptomycin 75 pg/ml and fungizone 8,7 )g/ml).

2. U.V. irradiation.

In a second stage, the cells were subjected to the action of ultraviolet rays. To this end, after 5 days, the culture medium defined in section 1 hereinabove was eliminated, the cellular tapetum was washed with a buffered sialine solution and then the cells were irradiated by UVA at 300 mJ/cm 2 Samples were then taken from the culture medium every day for 5 days.

It was then subjected to centrifugation at 1000 g for minutes, in order to separate the cells from the culture medium.

The medium or supernatant was evaporated down and a first active principle was obtained.

SThe cells separated by centrifugation were rinsed, scraped from S their vehicle auld then placed in suspension in distilled water in order to split them. The solution obtained after splitting constituted a second active principle.

The two active principles were then mixed in equal parts and the mixture was used in examples 2 to 9 hereinbelow to 12 illustrate the compositions according to the invention.

EXAMPLE 2 Sun cream The formulation is given in by weight: Active principle of Example 1 12 Liquid paraffin 34 Cocoa butter 2 Isopropyl myristate Beeswax 3 Magnesium lanolate 2.4 Lanolin alcohol 0.6 Polyethylene powder Antioxidant (butyl hydroxyanisole S+ butyl hydroxytoluene) 0.01 Sunscreen sold under the trade name "Parsol Ultra" by the company "Givaudan" 8 Perfume 1 Preservative 0.3 Denineralised water to 100 This composition protects the skin against the attack of U.V.

rays. It helps to limit the formation of free radicals and their consequences on the skin and it slows down the increavs in the microdepressionary system.

S EXAMPLE 3 Aqueous emulsion The formulation is given in by weight Active principle 113 Sorbitan mono st-a!,rate witk-h moles of ethylene oxide sold under the name "Tween 60"1 1 Cetyl alcohol Stearic acid 1.4 Triethanolamine 1.1 Mixture of carboxyvinyl acids sold under the name f'Carbopol 940"1 0.4 Karite oil liquid fraction 8 Wheat g~erm oil 6 Synthetic perhydrosqualene Antioxidant (butyl hydroxyanisole butyl hydroxytolUene) 0. 015 to Perfume 1 4 t Preservative 0.3 Demin~ ralised water to100 This oomposition, used in particular by people with dry skin, provides good hydration of the stratum corneum and protects the skin against significant variations in humidity. It therefore EXAE'LEIA--Skin.,care cream consisting of an oil emu,(sign The formulation is given in by weight: Active pr'inciple~ Sorbitan monisostearate Microcrystalline wax 1 Liquid wheat germr oil 7 Soya oil 7 Esters of f atty acids in C 8 -8 and of fatty alcohols in C 1

C

8 1 Organically modified montmorilJlonite gel and neutral oil gel (triglycerides of caprylic and capric acids) fJ Propylene alycol 3 Ii Antiox<idant, (butyl hydroxyanisole butyl hydrc.xytoluene) 0 .01 Preservative 0.3 Demineralised water to 100 Skin care creap consistingT of an oil emulsion I~ The formulation is given in by weight,.

ofActive picle(a) MagnEosium lanolate 14.4 Lanolin alcohol 3.6 Isopropyl myristate 8 Liquid paraffin i8 0* Sunflower oUt 22 Antioxidant (butyl hydroxyanisole butyl hydroxytoluene) 0.01 Ozokerite 4 Preservative wae o0.3 The active principle of these two emulsions wa, obtained from a culture of keratinocytes subjected to thermal. stress according to the following process: the keratinocytes w~q cultured at 37 C and at confluence the culture medium was replaced by a medium containing N'-2-hydroxyethylpiperazine-Nethane sulphonic acid. They were then placed in a thermostated water bath with stirring for 2 hours. This medium was then replaced by modified Eagle's medium containing foetal calf serum and then left in contact with the cells for 36 hours.

The two aqueous emulsions of Examples 4 and 5 protect the skin against the effects of dryness, in particular promoting false cell cohesion in the event of variations in temperature.

EXAMPLE 6 Tinted cream The formulation is given in by weight: oe fe Active ingredient 7 t.o' Partial glycerides of fatty acids 8 Cetyl alcohol Oleic acid decylester 8 Liquid paraffin 4 I Perhydrosqualene 16 Saturated fatty acid polyglycol ether 4 Magnesium and aluminium silicate 0.7 Polyethylene powder sold under the trade name "Polymist B6" by the company "Allied Chemicals" 4 Iron oxides 2.2 Preservative 0.3 Demineralised water to 100

AL

16 The active principle of this cream was obtained from a V culture of keratinocytes subjected to stress with the aid of a detergent.

SRegular application of this cream can improve skin colour, i which tends to dull with age, especially if the skin is exposed to a polluted environment.

EXAMPLE 7 Body milk Ij The formulation is given in by weight: Active principle Glycerol stearate 2 Sorbitan monostearate with 20 moles S. of ethylene oxide 1 S* Stearic acid 1.4 Triethanolamine 0.9 Mixture of carboxyvinyl acids sold under the trade name S"Carbopol 940" 0.2 Jojoba oil 3 Liquid paraffin S Antioxidant (butyl hydroxyanisole I butyl hydroxytoluene) 0.01 Perfume 1 Preservative 0.3 S" Demineralised water to 100 The active principle of this milk was obtained from a culture of fibroblasts subjected to stress by a deficiency of vitamins.

17 The milk, applied to the entire body, can reduce the effects of vitamin deficiency or sub-deficiency appearing in the course of ageing.

EXAMPLE 8 Skin care cream An aqueous dispersion of lipidic vesicles was manufactured by the process described in French Patent 2 315 991 from the following substances: Non-ionic amphiphilic lipid of general formula: i O CH. CH o I in which R is a hexadecyl radical and f has an average statistical value equal to 3 3.8 g V-sitosterol 3.8.g Dicetyl phosphate 0.4 g Preservative 0.3 g Demineralised water 27.6 g Active principle 20 g S* The following substances were added to the dispersion of vesicles obtained in the first phase: SSunflower oil 35 g Perfume 0.6 g Mixture of carboxyvinyl acids sold under the trade name "Carbopol 940" 0.2 g Triethanolamine 0.2 g Demineralised water 8.1 g The active principle of this cream was obtained from a culture of fibroblasts subjected to electromagnetic stress.

The culture medium was subjected to a magnetic field of gauss having a frequency of 50 Hertz, and having a peak with a duration of 5 milliseconds obtained with the aid of a pair of Helmholtz coils. The application time is 3 days.

The replenishing properties of this cream can lessen the signs of ageing, such as wrinkles.

EXAMPLE 9 Serum The formulation is given in by weight: i *i Hydroxypropylmethyl cellulose 0.3 Xanthan gum 0.2 Methyl glucose polyethylene glycol ether called methyl gluceth-20 in the "CTFA Cosmetic Di tionary" 1982 published by "The Cosmetic Toiletry and I Fragrance Association" 1 l Methyl parahydroxybenzoate 0.15 Propyl parahydroxybenzoate 0.05 Perfume q.s.

Active principle to 100 The active principle of this serum was obtained from a r 19 culture of keratinocytes and fibroblasts, subjected to simultaneous stress by vitamin deficiency and by ultraviolet rays.

i This composition improves skin tonicity.

In all of these examples of formulation, the active principle employed can be replaced by an active principle obtained from a i cell culture subjected to some other type of stress.

Ii i i e* 0

I

9 0L

Claims (14)

1. Cosmetic composition containing, in a cosmetically acceptable vehicle, at least one compound obtained by the culture of animal cells, characterised in that the said compound is a substance protecting the skin or the hair, synthesised by the said cells in response to at least one situation of stress during in vitro culture.

2. Composition according to claim 1, characterised in that the animal cells that produce the protective substance(s) are cells extracted from human skin, or from the shaft of a human hair or an eyelash.

3. Composition according to o<laim 2, characterised in that the cells from human skin are keratinocytes and/or fibroblasts. S•

4. Composition according to either of claims 2 and 3, characterlsed in that the cells producing the protective substance(s) are taken from the person intended to use the cosmetic composition. g 00 0

5. Composition according to claim 4, characterised in that the cells removed are frozen and kept for several years before being returned to culture.

6. Composition according to one of claims 1 to characterised in that the stress is selected from the group S formed by the action of ultraviolet rays, variations in temperature, an electromagnetic field, a chemical agent and/or a deficiency of an element normally present in the I I I i I 0** 9 5 SS** 9 9 21 culture medium.

7. Composition according to on of claims 1 to 6, characterised in that it contains active principles obtained in response to several stresses.

8. Composition according to one o£ claims 1 to 7, characterised in that the cells used are placed in culture, preferably until confluence, and are then subjected to the selected stress (or stresses).

9. Composition according to claim 8, characterised in that the culture is effected in the presence of precursors of the substances that will be synthesised as a result of the stress.

10. Composition according to one of claims 1 to 9, characterised in that the protective substances are used, without prior separation or purification, in the form of an active principle formed by the culture medium and/or the cells and/or a solution obtained after splitting the cells.

11. Composition according to claim 10, characterised in that the culture medium and/or the solution obtained by splitting the cells are concentrated.

12. Composition according to one of claims 1 to 11, characterised in that its cosmetically acceptable vehicle contains at least one cosmetically active compound.

13. Composition according to one of claims 1 to 12, characterised in that it is in the form of a cream, milk, lotion or serum.

14. Composition according to claim 13, in the form of a cream, characterised in that it contains from 0.1 to 30, by weight of the active principle(s) as defined in either of claims 10 and Composition according to claim 13, in the form of a serum, charact~irised in that it contains from 30 to 100 -0 by wel-ght of the active prin. -'le as defined in either of claims 10 and 11. DATED this 19th day of Apri- '990. .4.4 4. .4 4 9 4 4 S. 4' 4 .44 0 I I S 4 4 ~i .44. 4 L 1OREAL -ATERMARK PATENT TRADEMARK ATTORNEYS "THE ATRIUM" 290 BURWCOD ROAD HAWTHORN. VIC. 3122. k *Q 44 4 4 I o~tir, ~thh 3 n 2 5 O 2