AU638328B2 - Cosmetic or dermopharmaceutical composition containing vesicles consisting of a mixture of phospholipids/ glycolipids - Google Patents

Abstract

The invention relates to a cosmetic or dermopharmaceutical composition consisting of an aqueous dispersion of lipid vesicles. The lipids of the layers forming the walls of at least some of the vesicles contain 50 to 75 % of phospholipids and 50 to 25 % of glycolipids, including 1 to 5 % of gangliosides. These lipids may be extracted from the membranes of fat globules of a mammalian milk, in particular from buttermilk made from cow's milk.

Description

638328 COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952.69 COMPLETE SPECIFICATION (OR IGINAL) Class I Application Number: Lodged: Form nt. Class Complete Specification Lodged: Accepted: Published: Priority Related Art: 0 *0 Name of Applicant: Address of Applicant Actual Inventor: Address for Service L 'OREAL 14, rue Royale 75008 Paris, France ROSE-MARIE HANDJANI, ALAIN RIBIER and LADISLAS C0LAR0W WATERMARK PATENT TRADEMARK ATTORNEYS.

LOCKED BAG NO. 5, HAWTHORN, VICTORIA 3122, AUSTRLIA

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Complete Specification for the invention entitled: COSMETIC OR DERMOPHARMACEUTICAL COMPOSITION CONTAINING VESICLES CONSISTING OF A MIXTURE OF PHOSPHOLIPIDS/GLYCOLIPIDS The following statement is a full description of this invention, including the best method of performing it known to o 9 0 9* COSMETIC OR DERMOPHARMACEUTICAL COMPOSITION CONTAINING VESICLES CONSISTING OF A MIXTURE OF PHOSPHOLIPIDS/GLYCOLIPIDS This invention concerns a cosmetic or dermopharmaceutical composition containing lipidic vesicles dispersed in a continuous aqueous phase.

It is known that the use in cosmetic compositions of lipidic vesicles dispersed in a continuous aqueous phase has already long been suggestedj the said vesicles having leaf-like structures consisting of two or more lipidic layers'each separated by layers of aqueous phase; These vesicles may thus serve to encapsulate active watersoluble substances (see more particularly FR 2 315 991) in aqueous compartments between,lipidic layers. Furthermore the'use of these vesicles to.maintain.the stability of a dispersion in aqueous phase, of a liquid phase non-miscible with water, has been suggested (see FR 2 490 504). Consequently, cosmetic compositions have been proposed, with lipidic vesicles in aqueous dispersion on the one hand, and droplets of a phase non-miscible with water on the other hand, the S said droplets being able to enclose cosmetically active substances.

In the state of the art, amphiphilic lipids being able to form the vesicles of those compositions, either ionic lipids or non-ionic lipids have been proposed both types of vesicles may be mixed within a same composition.

To date and according to the particular aims in mind, certain S ionic or non-ionic lipids selected for their special behaviour has been suggested, and for each particular use additives to those amphiphilic vesicles such as cholesterol or dicetyl-phosphate, have been proposed for instance, their nature and quantity being selected according to the specific properties required for the vesicles in the relevant application.

S30 Vesicles for cosmetic or dermopharmaceutical purposes must essentially have four properties as outlined below: -firstly, the vesicles encapsulation capacity must be as large as possible, particularly with water-solubles solutes; -secondly, the vesicle size must be stable in relation to storage time and temperature, to ensure constant composition in storage; *e f r I -2- -thirdly, the vesicle composition must be chemically stable in relation to preservation time and temperature, particularly with regard to the perox ides content, so that the compositions may remain stable during storage; -fourthly, the vesicles must be easily tolerated by the skin, their constituent lipids more particularly having a nil or very low citotoxicity towards epidermis cells and derm.

Vesicles made with natural origin lipids and known additives have to date failed to display simultaneously the four qualities outlined above, and the choice of lipids and additives as well as their proportions have had to be adjusted to achieve a balance as satisfactory as possible allowing for the particular aim of the cosmetic application in each specific case. It was clearly most desirable to have a natural origin lipid, able on the one hand to S 15 allow the formation of vesicles, and by its nature, giving the said vesicles all of the four aforegoing qualities.

The purpose of the invention is a cosmetic or dermopharmaceutical composition containing vesicles simultaneously having the aforegoing four qualities, the level of each quality being of the same order of magnitude as the best for the relevant required quality in any one of the amphiphilic lipids of natural origin.known for forming vesicles. According to the invention, the use of complex lipids such as those in fatty globules of the milk of a mammal is proposed as vesicle amphiphilic lipids of natural origin. Surprisingly, it has been found that the use of these complex lipids in appropriate proportions, simultaneously allowed the best levels to be achieved for the aforesaid four qualities.

Milk from a mammal is known t6 contain in an aqueous medium, a high proportion of lipids, in addition to lactose and proteins. The lipids consist of two classes predominantly, on the one hand neutral lipids consisting particularly of triglycerides and sterols, and complex lipids in lesser quantity on the other hand, said complex lipids forming the components elements of milk fatty globule membranes. The membrane lipids come under two headings phospholipids on the one hand and glycolipids on the other.

3 The phospholipids are alcohol phosphorylated derivatives, i.e. glycerol (glycerophospholipid) or sphingosine (sphingophospholipid); in the first case both alcohol functions of non-phosphorylated glycerol are esterified with fatty acids; in the second case, the sphingosine amine function is connected by amide link with a fatty acid. In all cases the phospholipid molecules contain one or two fatty chains. In milk from mammals the phospholipid fatty chains are linear and mostly C 16

-C

22 The glycolipids structure derives from the structure of the sphingosine by bounding a fatty acid to the amine function by amide link, and by fixing one or several ose functions to the sphingosine primary hydroxyl group. In that class on fixing at least one osidic residue, either glucose or galactose, the cerebroside sub-class is obtained; glangliosides are ,btained if hexosamine and sialic residues are fixed in addition to osidic residues. The gangliosides are therefore a sub-class of glycolipids.

It has been found (and this forms the basis of the invention) that by extracting the complex lipids in membrane fatty globules such as found in mammal milk and using these complex lipids to form vesicles, this produces vesicles of the four best qualities required and at the best level. In this operation, relative phospholipid and glycolipid proportions of the milk extract, 20 are governed to some extent by the extraction process and raw material used. It has also been found according to the invention, that if complex lipids used to form the vesicles consist by weight of 50 to 75% of phospholipids and 50 to of glycolipids, of which at most 5% by weight of gangliosides, then regardless of original raw material(s), the aforesaid quality is obtained. The relevant vesicles are named 'lactovesicles' hereinafter regardless of the particular raw material(s) actually used.

:f European Patent application 89-118 183.6 describes the complex lipids separation from a lipidic mixture; it also describes, on the one hand the S: separation of phospholipids, and on the other hand the separation of glycolipids and finally of gangliosides. The technician can thus separate those three fractions and reconstitute the weight formulation indicated above, thereby obtaining regardless of original raw materials a complex lipids formulation- I f -4to allow the production of lactovesicles for the compositions according to the invention. More simply, it is also possible to select a raw material allowing by simple extraction of complex lipids, to obtain a satisfactory weight ratio of phospholipids to glycolipids, and a satisfactory weight ratio of gangliosides to total glycolipids. According to the invention a mammal milk is proposed as raw material, and more particularly derivatives of milk such as buttermilk, lactoserum or skimmed milk. Particularly interesting results were obtained using buttermilk from cow's milk as raw material.

It has been found that when the complex lipids in use contain polyunsaturated fatty chains, resultant vesicles offer further interesting advantages for cosmetic purposes. It is indeed known that certain human tissues, such as from brain and cornea, are 15 rich in polyunsaturated fatty acids; it has been found that the drop in polyunsaturated fatty acids content associated with ageing could be correlated with various functional diseases (ischemic cerebral illnesses, cataract). When complex lipids containing polyunsaturated fatty acids are processed as in the invention, to 20 form lactovesicles used in cosmetic or dermo-pharmaceutical products, beneficial effects connected with the role of these polyunsaturated fatty acids are obtained, particularly in epidermal homeostasis regulation and cellular fluidity and permeability, in regulation and inhibition of cutaneous inflammation.

25 In that field it has been found that the most interesting results are obtained when lactovesicles are formed from complex lipids, comprising a polyunsaturated fatty chains percentage between 15% and 25%, preferably close to 19% of the whole of the complex lipids fatty chains. This feature is naturally obtained 30 from complex lipids of fatty globule membranes from mammal's milk, particularly those extracted from cow's milk buttermilk.

It was also found that gangliosides present among complex lipid glycolipids used to form vesicles in compositions according to the invention, also favour obtaining the simultaneous vesicle qualities of the said compositions. The gangliosides indeed allow an electro-negative charge input (zeta potential -50+ 14 millivolts) into the vesicles, sufficient to ensure stability by inhibiting flocculation; the charge is nevertheless masked by the presence of osidic residues carried by the molecule at the end of the sphingosine skeleton, which limits interactions with other charged molecules such as proteins. This gangliosides effect was brought out by the half-life extension of liposomes in the blood flow see the paper by J Senior and G Gregoriadis presented to the 'Liposomes Research Days' at Florida University Convention (Gainefville USA) (28.02 02.03.1990).

The object of the present invention is thus a cosmetic or dermopharmaceutical composition in the form of aqueous dispersion -of vesicles limited by walls consisting of organised lipidic molecular layers, characterised by the fact that 90% by weight at least of wall .layers of at least some dispersion vesicles, known as 'lactovesicles', consist of 'complex lipids with by weight 50% to 75% of phospholipids and 50% to 25% of glycolipids, of which at most 5% consist of gangliosides, the above percentages by weight o being given in relation to the total weight of complex lipids, the S eventual complement consisting of lipo-soluble products.

Extraction of complex lipids from a natural raw material, 20 derived more particularly from milk, is achieved by any known process, for instance as described in EP-A-365868; extraction is also possible with acetone or supercritical carbonic acid, then followed by an enzymatic hydrolysis stage.

In a preferred embodiment, gangliosides represent 1 to 5 by 25 weight related to total weight of lactovesicles complex lipids; the lactovesicles complex lipids of the composition according to the invention, are those of which the mixture is found in the membranes of fatty globules of mammal milk; such complex lipids may be extracted from buttermilk, lactoserum or skimmed milk. It 30 is preferable for the weight of phospholipids in lactovesicles to Sbe substantially equal to twice the weight of glycolipids of the total whole of the lactovesicles complex lipids, gangliosides will usefully amount to approximately 3% of the weight.

It has been found advantageous for complex lipids fatty chains in lactovesicles to contain 15% to 25%, and preferably 19% approximately of polyunsaturated chains.

1 I -6t was found that excellent results were obtained using lactovesicles of which the complex lipids are obtained by direct extraction from cow's milk buttermilk.

In compositions according to the invention, the lactovesicles average diameter is advantageously between 10 and 1,000 nm. The quantity of lactovesicles present in a composition according to the invention may vary within wide limits, but compared with the total weight of composition there will generally be 0.1 to 20% by weight of complex lipids forming the lactovesicle walls.

The lactovesicles of compositions according to the invention are prepared by one of the known methods for obtaining a vesicle aqueous dispersion from amphiphilic lipids the process claimed uhder FR-2 315 991 or operating procedures described in FR-2 221 122 may be applied in particular.

The composition according to the invention may also contain at least one class of vesicles other than lactovesicles, consisting of ionic lipids or non-ionic lipids or of a mixture of ionic and non-ionic lipids, the total vesicle lipids of the composition being at most 25% of the composition total weight. The additional 20 vesicles will preferably be of diameters between 10 and 1,000 nm.

Additional vesicles can be prepared separately in that case and the corresponding aqueous dispersion be mixed with that containing lactovesicles to obtain the composition according to the invention.

25 The composition according to the invention may also contain 1% to 40% by weight of at least one non-water-miscible dispersed phase, the proportion by weight of all vesicle lipids in relation to the dispersed (phase(s) being between 0,2:1 and 1:1; droplets of dispersed phase(s) should preferably be of an average diameter between 100 and 10,000 nm.

To form the above non-water-miscible phase dispersed in the

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*I form of droplets as outlined above, one compound at least may be used from the group consisting bf hydrocarbons, halogenated carbides, polysiloxanes, polydimethylsiloxanes, inorganic or organic acid esters, ethers and polyethers. Hexadecane and paraffin oil can be named among the hydrocarbons group, and perfluorotributylamine and perfluoro-decahydronaphthalene, fluoro-carbons, fluoroesters, fluoroethers and fluorosilicones among the halogenated carbides.

t -7- Where the non-water-miscible dispersed phase is a fatty acid and polyol ester, it can be usefully selected among the liquid triglycerides and esters of fatty acid and branch-chained alcohol as in the formula: R-COO-R' wherein R is the rest of fatty acid with 8 to 20 carbon atoms, and R' is a branch-chained hydrocarbon with 3 to 20 carbon atoms, or in which R is the rest of unsaturated fatty acid in C2 0

-C

22 and R' the rest of a fatty alcohol. Among the esters of fatty acid and polyol, mention should be made of oils of sunflower seed, maize soya, gourd, grape-seed, macadamia hazel-nut, fish and glycerol tricaprocaprylate. Among esters of higher fatty acids and branch-chained alcohol, purselane oil can be named.

Among esters of unsaturated fatty'acids'and fatty alcohol, jojoba oil should be noted.

Where the composition .according to the invention contains droplets of non-water-miscible dispersed phase, said composition S*o. may be prepared in a known manner in two steps: firstly, the e aqueous dispersion of vesicles is prepared, whether it only contains lactovesicles or additional vesicles as well; secondly, the non-water-miscible phase is added to the dispersion and 20 dispersed by shaking.

The composition as in the invention may contain at least a cosmetic or dermopharmaceutical active substance.In a first version the active substance is stored in vesicles of the composition, and protected by the vesicle walls. The active substance may be water- 25 soluble, and may thus be held in solution in the aqueous phase encapsulated in the vesicles of the compo.sition. However, the said active substance may also be liposoluble: it is then stored in the lactovesicle lipidic walls. If it contains additional vesicles other than the lactovesicles, the active substance may be stored under 30 the same conditions in these additional vesicles however, if the composition holds dispersed non-water-miscible phase, the active substance may also be stored in dispersed phase droplets. Where it is water-soluble, the active substance may also be contained in the continuous aqueous phase of the composition according to the invention.

Incorporation of the active substances in aqueous dispersions containing vesicles has already been described in the state of the art, for instance in FR-2 485 921.

-8- 'Among the active liposoluble substances should be quoted solar filters such 2-ethylhexyl paradimethylaminobenzoate, substances intended to improve dry skin or senile conditions, particularly non-saponifiables such as soya, rice,karite and avocado non-saponifiables, tocopherols, tocopherol nicotinate, vitamins E, F or A and their esters,retinoic acid, anti-oxidisers, essential fatty acids, glycyrrhetinic acid, keratolitics, carotenoids, -carotene, '-orizanol, ceramides and stearyl glycyrrhetinate.

Among active water soluble substances, moisteners such as glycerin, sorbitol, pentaerythritol, inositol, carboxylic pyrrolidone-acid and its salts, artificial suntan agents, such as dihydroxyacetone, erythrulose, glyceraldehyde, '-dialdehydes, such as tartaric aldehydes, skin colouring agents, solar filters, anti-perspirants, .deodorants, astringents, freshening-, tonichealing-, keratolytic-, depilatory -products, animal or vegetable tissue extracts, amniotic fluid, polysaccharides, anti-seborrehic agents, oxidising agents, such as hydrogen peroxide, or reducers such as thioglycolic acid and its salts. In dermopharmaceuticals mention can also be made as active substances of water-solubles, 20 vitamins, hormones, enzymes, vaccines, anti-inflammatories, antibiotics, bLctericides, selenium derivatives, hydroxyacids such as glycolic acid, nicotinic esters, glycerol, free radical captors, depigmentisers, and slimming preparations.

In its continuous phase, the composition according to the 25 invention may contain, at least an additive from the group of gelling-, alkalinisation-. or acidification- agents, preservers, colorants, opacifiers and perfumes. If the composition includes a non water-miscible phase dispersed as droplets, the additive is preferably added to the dispersion at the same time as the non water-miscible phase. The gelling agent may be added in a concentration between 0.1 and 2% by weight related to total weight of composition. As admissible gelling agents, cellulose derivatives such as hydroxyethylcellulose; synthetic polymers; algae derivatives, such as satiagum or even natural gums such as adracanth can be named. A mixture of carboxyvinylic acids commercially available under the name CARBOPOL 940, sold by Goodrich can be used advantageously.

The pH of the compositions according to the invention is preferably between 4 and 9.

For easier understanding of the invention, several embodiments are now described in -the examples below, purely as an illustration of non restrictive examples.

Example 1 Complex lipids preparation forming lactovesicle walls Sweet buttermilk from cow's milk is used as raw material. The buttermilk is decaseined and delactosed by diafiltration in a known manner; it is then spray-dried. This produces a buttermilk powder of the following composition: Ingredients Moisture (stoved 2 hours at 102°C). 3.2 Total solids 96.8 15 whereof: total lipids (Mojonnier method) 61.24 Proteins (N x 6.38) 32.43 Lactose (enzymatic determination) 1.24 Ash 1.87 100lOOg of this powder are homogenised in two stages, each time 20 with 500 ml of hexane/methanol mixture (94:6 by volume) for one minute at 50 0 C in a colloids mill. The homogenised product is then centrifuged each time for two minutes at 1500 g. The supernatant is then concentrated under vacuum until dry at 65 0 C. Total lipids are thus obtained, which remain fluid owing to their high butter 25 oil content.

In a second stage, complex lipids are separated from total lipids. To that end the total lipids obtained are cooled to 40 0

C,

homogenised in a colloids mill with 110 ml of.acetone, left to reach ambient temperature (23 0 C) and centrifuged for one minute at 30 2000 g. The clear supernatant with most of the neutral lipids and odorant compounds is then eliminated; the solid residue from centrifugation contains complex lipids. The solid residue is again dissolved in hexane and concentrated by vacuum evaporation. This produces material non-soluble in acetone; after taking up into suspension in three times its volume of hexane, complex lipids (approx. 80%) are separated from neutral lipids (approx. 20%) by absorption liquid chromatography with 11 g of treated silica gel.

The silica mesh-size is the fraction retained between sieves of 27 and 91 meshes/cm; it is available commercially among others from Merck, pre-treatment being stoving at 165 0 C for 16 hours.

During chromatography neutral lipids are adsorbed onto the silica.

while complex lipids forming reverse micelles, are not adsorbed and can thus be elutriated.

Complex lipids thus obtained (17.5 g) are homogenised in times their volume'of distilled water and the emulsion is then freeze-dried.

The presence in the complex lipids thus obtained of 66% of phospholipids and 33% of glycolipids is determined, among which 3% of gangliosidesi all these percentages being expressed in relation to complex lipids total weight.

The unsaturated fatty chains percentage is defined according to the whole of'the fatty chains for each chain length of phospholipids and glycolipids forming the complex lipids thus obtained.

The results of the determination are shown below: o*• 0

S

0 0 *0 0 0*0 OC o 0 0 00 0 0' *00 *0 0 4 0 *0 060 00 0 0 0 0 00 0 0 S S 0 0 0 0 *00 *0 0 *0 0 *0 0 Unsaturated fatty chains percentage related to I ~~~total -category fatty chains Type of lii C, Origin: Buttermilk i unsaturations number in unsaturated chain -12- It will he seen that of the complex lipids thus obtained, some 19% of phospholipids and glycolipids are unsaturated fatty chains, the unsaturations being c0-3 and J-6 (where presents the first double link position from the methyl end).

Example 2 Lactovesicles properties testing Lactovesicles are prepared from complex lipids obtained by way of example 1, using the process claimed in FR-2 315 991. The same procedure is adopted for comparative amphiphilic lipids.

Table I below compares on the one hand, encapsulation capacity of various tested lipids and on the other hand, the maintenance in time of vesicles encapsulation. Tested aqueous dispersion vesicles have an approximate mean diameter of 1,500 nm. The encapsulation rate is defined by the swelling rate in microlitres/mg: it shows the microlitres encapsulated in vesicles using 1 mg of lipids. Of the various tested amphiphilic lipids, the example 1 component lipids appear to have the best encapsulation capacity.

A study of vesicle capacity maintaining their encapsulation in time, can be deducted from leakage values after I hour, one day,eight days and thirty days. The leakage rate is determined by *20 encapsulating in the vesicles a 54 g/l glucose solution, measuring the glucose passing into the continuous aqueous phase after filtering vesicles through a SEPHADEX G 50 chromatographic gel column. Once more it appears that the quality of results for complex lipids in example 1 is comparable..with the best in the table (corresponding with total beef brain lipids extracts).

Dimensional stability in time of small vesicles obtained with the VIRTIS-type ultra-disperser has been studied. The left-hand column of Table II shows the initial size of tested vesicles. The 4 vesicle-sizes were checked after one and two months in storage; storage temperature was maintained at 9 4, 20, 37 or 45°C. It will be noted that for Example 1 complex lipids, the results show perfect dimensional stability and are thus at the quality level of the best results obtained with other tested lipids. Lactovesicles of the composition according to the invention thus maintain a perfectly stable size in time.

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TABLE I LIPID COMPOSITION SWELLING LEAKAGE RATE AS after..

VESICLE LIPID IN by weight) fl/mg 1 hr 1 day 8 days 30 days Origin Commercial name Supplier PL GL IG I CH SOYA EPIKURON 200 LUCAS METER >97 0 0 0 0.9 52 45 100 170 >96 0 0 0 1.0 69 91 100 145 85 0 0 0 0.3 89 100 100 65 0 0 0 0.6 o o60 0 EGG OVOCITHIN 200 u~cs METER >92 0 0 0 0 to00 160 >85 0 0 0 1.7 so60 65 100 BUTTER -MILKUTTER TOTAL LIPIDS NESTCc 18 9 1 0.3 65 100 EXAMPLE I

COMPLEX

LIPIDS 1 66 33 3 0 7.4 10 21 43 73 '*BEEF BRAIN INIEO 9 76 15 2.2 22 23 GLYCOLIPIDS TOTAL BEEF BRAIN LIPIDS EXTRACT (RHC powder) PEnTAPHARM 44 35 20 6.4 21 36 70 83

BEEF

MARROW CHOLECITHIN GIVAUDrA 39 20 12 1.1 26 21 32 PL Phospholipds GL Glycolipids GG gangliosides CH Cholesterol *The encapsulation capacity of the first material is lower than that of buttermilk, in spite of the presence in the lst named of cholesterol, a lipid well-known for optimising this property.

gangliosides %age included in glycosides %age 3 Nt. 0 0) 3 .5 3)00* 0* *0 0 0 09 0 a me 0 00 S 0 0.) 4 0B 0 5 0 OS 0 600 TABLE II Vesicles average diameter 10 nm VESICLE LIPID Time T 0 T 1 month T 2 months 8 in OC 4 20 37 45 4 20 37 Origin Commercial name Supplier SOYA EPIKURON 200 0,44 F F F F F F T F 170 LUCAS 0,14 0,16 0,16 0,15 0,21 0,14 10. 14 0,23 0,43

MEYER

145 0,15 0,15 0,15 20 0,20 0,15 0,18 0.18" 0,17 100 0,13 0,13 0.13 0,14 0,14 0,13 0.13 C C

ECG

OVOCITHIN 200 LUCAS 0,.31 F F F F F F F F MEYER 0,17 0,17 0,18 0,19 0,21 0,18 0,18 0,20 0,39 RUTTER- Example 1

MILK

complex lipids 0,25 0.25 0,25 0.24 0,23 0,24 0.24 0,23 0.22 BEEF BRAIN GLYCOLIPIDS.

NIKKO 0.32 0,39 0,41 0,44 0.42 0,45 0.45 0,44 0,44 TOTAL BEEF BRAIN LIPIDS EXTRACT (RC powder) PENAPHAF 0,26 0,25 0,24 0,24 0,24 0,26 0,25 0,25 0,25 EXTRACT (RHC powder) BEEF MARROW CHOLECITHIN GIVAUDAN 0.18 0,21 0,20 0,21 0.22 0,2 0.2 0,2 0,22 Numerous non-lamellar fatty globules indicatinq a chemical change F flocculation, fusion C split into two phases (aqueous and fatty) 15 a a a.

a Table III shows vesicle chemical stability results according to preservation time and temperature. To study chemical stability, the peroxides content of compositions was checked initially, after onethen two months in storage, It is known that polyunsaturated fatty acids auto-oxidation is accompanied by increased absorption for a wavelength of 233/234 nm. The increase is proportional to the peroxides thus formed (see J.A.O.C.S. Volume 63, N o 7, p. 883).

(July 1986). Absorption was therefore measured with a spectro photometer at this wavelength. Absorption A is expressed as a percentage .by the ratio A/o 100 I 100, wherein I and Io are respectively the transmitted and Oincident fluxes it is referenced in milliunits of optical density OD (OD log Table III shows the absorption values in milliunits for the wavelength under consideration. The vesicle dispersions subjected to these tests were obtained -4 with freeze-dried vesicles dispersed at the rate of 10 g/ml in methanol. When interpreting the table, absorption evolution between time 0 and the test-time should be considered for a given vesicle lipid, and not the absolute absorption value. Where the evolution in time is extensive, the composition is deemed unstable Table III records instability, the unstable results being underlined.

It is found that Example I complex lipids chemical stability in time is of the same quality order as the best results obtained for comparative vesicle lipids.

Finally to investigate tolerance by the skin and citotoxicity with 25 regard to epidermis and de:-m cells, tests were carried out on fibroblasts and keratinocytes. These tests results are recorded in Table IV.

Fibroblasts isolated from a human skin fragment were produced in a minimum essential culture medium (MEM) supplied by GIBCO, with a 10% addition of foetal calf serum; they were kept in a moist atmosphere at 37 0 C with 5% CO 2 Cells were replanted each week and sown at constant density (106 cells/15 ml of culture). Fibroblasts from a 5th day culture were used for the experiments.

Keratinocytes were also isolated from a human skin fragment.

Cells were grown in MCDB 153 culture medium (mildly calcitic 0,1 mM CaC12) supplied by CLONETICS, with added epidermal growth factor, insulin, phosphoethanolamine, ethanolamine *0 a a a

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see, e00 6. 59 S *5

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-16and bovine pituitary extracts (proteins 70 mg/l of medium); the cells were kept in a moist atmosphere at 37 0 C with 5% C0 2 The cells were replanted running together and grown at constant density (105 cells 10 ml of culture). The keratinocytes were used in the running together condition for the experiment.

Lipids tested on the cell cultures were made available in the form of vesicle dispersions; six concentrations, between 1 mg/ml and 10 3 mg/ml were used for the tests. The 2 h 30 cytotoxicity study .featured the MEM dispersion medium for fibroblasts and MCDB 153 medium for keratinocytes. The 48 hour cytotoxicity tests featured fibroblasts in MEM dispersion medium with 10% of calf foetal serum, and keratinocytes in whole MCDB medium (with bovine pituitary extracts).

Under the above culture conditions, cells were started after trypsination in treated microplate hollows for cell culture, at the rate of 20,000 cells per hollow. The cells were grown 48 hours in humid atmosphere as quoted above. In each case the study features 5 micro-hollows and toxicity is assessed for two tests.

Cell viability was checked by colorimetric method based on 20 cellular mitochondrial metabolism of a tetrazolium salt (viz. 3- 2,5-diphenyl) tetrazolium bromide (Mosmann test). The pale yellow substrate is tranformed by living cells into a dark blue metabolite, during the reaction. The metabolite absorption is directly proportional to,. cell density. Optical 25 density was determined at 570 nanometres from the micro-plates by Elisa (Biotech EL 308) spectrophotometer; as stated, the optical density is the relationship log (incident/transmitted flux). The toxicity of test-lipids dispersions is assessed by relating optical densities against a control treated under the same conditions. The results are expressed as a toxicity percentage.

The relevant lethal dose 50 (LD 50) is calculated by simple regression in the case of high toxicity. The methodology is fully detailed in 'Toxicity in Vitro' Vol.3, N 0 2, p.103-109, 1989.

Table IV summarises details to be drawn from the tests. It is found in particular, that the cytotoxicity of Example 1 complex lipids is clearly lower that that of soya lecithins used in comparison.

S 0 0 0 -17- -ll- This group of tests thus shows that of the four qualities required of vesicles intended for cosmetic purposes, Example 1 complex lipids are at the highest level found for any of the various vesicle lipids tested in comparison; each comparative lipid has a poor behaviour with reference to at least one of the required qualities.

Example 3 Skin-firming liposerumusing lactovesicles Using a 300 ml glass flaski 0.5 g of exemple 1 complex lipids are weighed offj then 6 g of demineralised water are addedi the 0' the mixture being allowed to swell for 1 hour at 40 0 C. The mixture is then stirred with a spatula; an addition is then made of 5 g of glycerin, 1 g of hydroxyproli.ne.and 24.5 g of aqueous solution obtained by crushing animal placenta tissue, available under the commercial name of PHYLDERM from GATTEFOSSE. The mixture is processed at ambient temperature, in a Virtis type ultra-disperser until the average size of vesicle obtained is 200 nm or less. The size is checked in a COULTRONICS NANOSILER quasi-elastic light diffusion granulometer.

0.1 g of sodium hyaluronate dispersed and preexpanded in 9.9 g of demineralised water is added to the dispersion.

The following are then added to the dispersion thus obtained: 0.2 g carboxyvinylic acids mixture available from Goodrich under the name CARBOPOL 0.25 triethanolamine.............. 0.25 g methyl 0.3 g demineralised water 52 g This gives a beige-coloured liquid serum, which, is placed in a flask with hand-metering pump. The serum is used in twice-daily topic applications each of approximately 2.5 mg/cm 2 After 3 weeks of use, a distinct firming of the skin is noted.

Example 4 Irritated skin care-cream using lactovesicles Using a 300 ml glass flask, 3 g of complex lipids obtained from example 1 are weighed off, then 0.15 g of(-tocopherol are added. The two products are mixed with a spatula. A 27 g addition of demineralised water is made, the mixture being left to swell for 1 hour at 40 0

C.

S S S *5 5 .5 S 55

S

5* *5 *OS S S 55 S S S S S S S S S S 55 TABLE III Absorption in milliunits at 233 nm VESICLE LIPID T T 0 T 1 month T= 2 months 6 OC 4 20 37 5 4 20 127 Origin Commercial name Supplier

SOYA

EPIKURON 200 LUCAS 104 96 117 172 125 135 118 168 182 170 MEYER 60 93 97 101 92 106 116 113 83 145 87 126 76 72 72 124 '140 140 137 100 108 135 175 156 337 575 773 917

EGG

OVOTHIN 200 LUCAS 80 68 162 94 140 189 29 268 265 160 MEYER 69 68 64 56 73 174 180 243 234 BUTTER- EXAMPLE 1 MILK COMPLEX LIPIDS 117 12 1 112* MILK COMPLEX LIPIDS 117 121 123 128 131 112 127 131 143 BEEF BRAIN GLYCOLIPIDS NIKKO 52 46 60 88 102 50 67 105 112 LIPID TOTAL EXTRACTS OF BEEF BRAIN PENTAPHARM 94 104 20A 129 223 149 232 34 207 BEEF BONE MARROW CHOLECITHINE GIVAUDAN 97 104 102 113 142 101 109 112 140 1 S S S

S.

S

Sc 0 S 55 S 0 e. S S 0*.

TABLE

S 55 5 S S S S S S S S S S S

TOXICITY

VESICLE

LIPI D FIBRO0BLAST'S

KERATINOCYTS

48 H SOYA LECITHIN Lipold PC 9 PE SOYA -LECITHIN Van de Moortelle at 53 PC SOYA LECITHIN Van de Moortelle at 38 t- PE HT NT DL50 at 5 10-3g/m1 SOYA LECITHIN Van de Moortelle.

at 50 PI COMPLEX LIPIDS -of exampleI NT non toxic -19- The mixture is again stirred, adding 3 g of glycerin, 0.5 g of allantoine and 20 g of 1% aqueous solution of monomethyltrisinalol lactate (available from EXSYMOL under the name LASILIUM).

Using the same technique as in example 3, the mixture is processed in a Virtis-type ultra-disperser until the average size of vesicle obtained is 200 nm or less.

A 15 g addition of black currant oil is made to the dispersion with 0.5 g of solar filter available from BASF under the name UVINUL M 40, 0.5 g of solar filter available from GIVAUDAN under the name PARSOL M C X and finally 0.1 g of perfume. The whole mixture is processed at ambient temperature in a Virtis-type ultra-disperser until the oil globules mean size is less tnan 500 nm.

The following are then added to the dispersion thus obtained: carboxyvinylic acids mixture available from Goodrich under the name CARBOPOL 940 0,4 g triethanolamine. 0,4 g methyl parahydroxybenzoate 0,3 g demineralised water 29,6 g This produces a broken-white-coloured cream with a viscosity 20 of 20 poises. The cream is applied to acne-prone greasy skins each morning for a period of 3 weeks, at the rate of 3 mg/cm 2 per application. It is found that this cream allows skin-irritation of the subject to be reduced.

Example 5 Day cream for dry-skin care using lactovesicles 25 Using a 100 ml glass flask, 1 g exemple 1 complex lipids are weighed off, then 5 g of demineralised water are added, leaving the mixture to swell for 1 hour at 40°C. The mixture is then stirred with a spatula, adding 1 g of hydroxyproline and 10 g of 1% by weight aqueous solution of monomethyltrisilanol mannuronate available from EXSYMOL under the commercial name ALGISIUM.

The whole mixture is processed at ambient temperature in a Virtistype ultra-disperser until the average size of vesicles obtained is less than 200 nm using the technique defined in Example 3. This provides a first vesicle phase.

The following products are placed in a glass 300 ml flask: non-ionic amphiphilic lipid, according to the formula: R 40CH 2

S.

.S

in which formula R is an hexadecyl radical and n is a statistical value equal to 3 0.95 g cholesterol .0.95 g dicetyl phosphate 0.10 g The three products are mixed by melting at 110 0 C under nitrogen cover,then bringing the temperature of the melt back to 90°C. An addition is then made of 3 g of glycerine dissolved in 20 g of demineralised water. At 70 0 C, the whole is placed for processing in a Virtis-type ultra-disperser until the average size of vesicles obtained is less than 300 nm with the technique shown in Example 3. This second vesicle phase is left to return to ambient temperature.

The first vesicle phase is then poured into the second phase.

The following products are then added to the mixture: macadamia oil .14 g volatile silicone oil 10 g solar filter from BASF sold as UVINUL M 40 0.5 g solar filter from GIVAUDAN sold as PARSOL MCX 0.5 g perfume 0.5 g At a 15°C thermostat-controlled temperature, the whole is placed for processing in a Virtis-type ultra-disperser until the average size of oil globules obtained is less than 500 nm.

The following products are then added to the mixture: carboxyvinylic acids mixture available from Goodrich under the name CARBOPOL 940 0.42 g triethanolamine 0.42 g methyl parahydroxybenzoate 0.3 g demineralised water 31.36 g The product is a white cream with a viscosity of 30 poises.

This cream is applied to dry skins each morning for 3 weeks at the rate of approximately 2 mg/cm 2 per application. A considerable reduction of skin-dryness is noted at the end of the period.

2-5 S*

Claims (17)

1. Cosmetic or dermopharmaceutical composition consisting of an aqueous dispersion of vesicles having walls consisting of lipidic molecular layers, characterised in that at least 90 by weight of wall layers of at least some dispersion vesicles, known as "lactovesicles", consist of complex lipids comprising 50 to 75 by weight of phospholipids and 50 to 25 by weight of glycolipids, of which at most 5 by weight consist of gangliosides, and balance consisting of liposoluble products, the above percentages being given in relation to the total weight of complex lipids.

2. Composition according to claim 1, characterised in that the lactovesicle complex lipids comprise 1 to 5 by weight of gangliosides.

3. Composition according to claim 2, characterised in that the lactovesicle complex lipids comprise approximately 3 by weight of gangliosides.

4. Composition according to one of claims 1 to 3, characterised in that the composition lactovesicle complex lipids are those of which the mixture is found in the membranes of fatty globules in the mammal milk. Composition according to claim 4, characterised in that the lactovesicle complex lipids are extracted from buttermilk, lactoserum or skimmed milk.

6. Composition according to one of claims 1 to 5, characterised in that the weight of phospholipids in lactovesicle complex lipids is substantially equal to twice the weight of glycolipids.

7. Composition according to one of claims 1 to 6, characterised in that the fatty chains of lactovesicle complex lipids comprise 15 to 25 of polyunsaturated chains.

8. Composition according to claim 7, characterised in that the lactovesicle complex lipids fatty chains comprise approximately 19 of polyunsaturated chains.

9. Composition according to claim 3, characterised in that the weight of phospholipids in lactovesicle complex lipids is substentially equal to twice the weight of glycolipids and the lactovesicle complex lipids fatty chains comprise approximately 19% of Spolyunsaturated chains and further characterised in that the lactovesicle complex lipids are obtained by direct extraction from cow's milk buttermilk. oe Composition according to one of claims 1 to 9, characterised in that the mean diameter of lactovesicles ranges between 10 and 1,000nm.

11. Composition according to one of claims 1 to 10, characterised in that it contains 0.1 to 20 by weight of complex lipids forming the wall of lactovesicles as related to composition total weight.

12. Composition according to one of claims 1 to 11, characterised in that it contains at least one class of additional vesicles, other than lactovesicles, consisting of ionic or non ionic lipids or mixtures of ionic and non-ionic lipids the total vesicle lipids being at most equal to 25 of the composition total weight.

13. Composition according to claim 12, characterised in that additional vesicles have a mean diameter between 10 and 1,000 nm.

14. Composition according to one of claims 1 to 13, characterised in that it contains 1 to 40 by weight of at least one non-water-miscible dispersed phase, the weight proportion of vesicle lipids related to all dispersed phase(s) being between 0.2:1 and 1:1. Composition according to claim 14, characterised in that the droplets of the dispersed non-water-miscible phase have an average diameter between 100 and 10,000 nm.

16. Composition according to one of claims 14 or 15, characterised in that the dispersed non-water-miscible phase is made up of at least one compound of the group consisting of hydrocarbons, halogenated o* RA cS 9 o* o• *o*o *o i I I -23- carbices, polysiloxanes, polydimethylsiloxanes, organic or inorganic acid esters, ethers and polyeth- ers. 17/ Composition according to claim 16, character- ised in that the dispersed non-water-miscible phase includes at least one compound of the group comprising hexadecane, paraffin oil, perfluorobutylamine, per- fluorodecahydronaphthalene, fluorocarbons, fluoroes- ters, fluoroethers and fluorosilicones. 18/ Composition according to claim 16, character- ised in that the dispersed non-water-miscible phase is an oil selected from the group consisting of esters of fatty acids and polyol and branch-chained alcohol of formula R-COO-R', wherein R represents the rest of 15 fatty acid with 8 to 20 carbon atoms and R' a branch- chained hydrocarbon with 3 to 20 carbon atoms. 19/ Composition according to claim 18, character- ised in that the dispersed non-water-miscible phase comprises at least one compound of the group of oils 20 of sunflower seed, maize, soya, gourd, grapeseed, jojoba, macadamia, purselane and glycerol tricapro- caprylate. 20/ Composition according to one of claims 1 to 19, characterised in that it contains at least one active substance of cosmetic or dermopharmaceutical nature. 21/ Composition according to claim 20, character- S* ised in that the active substance is stored in the vesicles of the composition. 22/ Composition according to claim 21, character- ised in that the active substance is soluble in water and encapsulated within the vesicles of the composi- tion. 23/ Composition according to claim 20, character- ised in that the active substance is liposoluble. 24/ Composition according to claim 24 characterised in that the active substance is stored within the walls of the composition. Composition according to any one of claims 14 to 19, characterised in that the composition contains at least one liposoluble active substance of cosmetic or dermopharmaceutical nature and that the active substance is stored within the droplets of the non-water miscible dispersed phase.

26. Composition according to claim 20, characterised in that its continuous aqueous phase contains at least one active water-soluble substance.

27. Composition according to one of claims 22 or 26, characterised in that the active water-soluble substance is selected from the group consisting of moisteners, agents for artificial sun-tan, agents for colouring, water soluble solar filters, perspiration agents, deodorants, astringents, freshening-, tonic-, healing-, keratolytic products, depilatories, animal or vegetable tissue extracts, amniotic fluid, polysaccharides, antiseborrhoeics, oxidising and reducing agents, vitamins, hormones, enzymes, vaccines, anti- inflammatories, antibiotics, bactericides, selenium derivatives, hydroxyacids, glycerol, nicotonic esters, free radical captors, depigmentisers and slimming products.

28. Composition according to claim 23, characterised in that the active liposoluble substance is selected from the group consisting of liposoluble solar filters, substances intended to improve dry or senile skin condition, soya, rice, karite, and avocado non-saponifiable, tocopherols, tocopherol nicotinate, vitamins E, F or A and their esters, retinoic acid, antioxidisers, essential fatty acids, glycyrrhetinic acid, keratolytics, carotenoids, 13-carotene, y-orizanol, ceramides and stearyl glycyrrhetinate.

29. Composition according to one of claims 1 to 28, characterised in that its continuous aqueous active phase contains at least one water soluble e• *0 co I I, S. additive selected from the group consisting of gelling-, alkalinisation- and/or acidification agents, preservers, colorants, opacifiers and perfumes. Composition according to claim 29, character- ised in that it contains by weight related to total composition weight, 0.1 to 2 of at least one gelling agent selected from the group of cellulose deriva- tives, synthetic polymers, algae derivatives and natural gums. 9 DATED this 26th day of April 1991. L'OREAL WATERMARK PATENT TRADEMARK ATTORNEYS "THE ATRIUM" 290 BURWOOD ROAD HAWTHORN. VIC. 3122. 1 ee Se