WO2000040611A1

WO2000040611A1 - Slimming cosmetic compositions

Info

Publication number: WO2000040611A1
Application number: PCT/FR1999/003110
Authority: WO
Inventors: Karl Lintner
Original assignee: Sederma S.A.
Priority date: 1998-12-30
Filing date: 1999-12-09
Publication date: 2000-07-13
Also published as: FR2788058B1; FR2788058A1; AU1569800A

Abstract

The invention relates to peptide sequences derived from the parathyroid hormone (pTh), whereby said sequences exhibit lipolytic activity that is used to advantageous effect in cosmetic or dermatological slimming products that are administered topically. Said activity is enhanced when the peptides are chemically modified so as to increase their lipophilicity. The peptides can be obtained by means of synthesis, biotechnology or by moderate hydrolysis of plant proteins.

Description

TITLE Slimming cosmetic compositions

The cosmetic industry is constantly looking for new active ingredients with lipolytic activities, to integrate them into so-called slimming products. Many substances (pure molecules such as 5 xanthine derivatives; complex mixtures such as certain plant extracts, etc.) are proposed and used.

The market nevertheless demands new and ever more active products. Adrenergic substances (adrenaline and the like) are well known for their impressive capacity to stimulate lipolysis in adipocytes, but their use is strictly prohibited in cosmetics and dermopharmaceucy.

Recently, other classes of substances, of different nature, have been identified as also capable of stimulating, to varying degrees, the lipolysis of triglycerides in human and / or animal adipocytes. These are short chain peptides of a hormonal nature such as, for example,

Para-thyroid hormone (pTH (1-84)) or its fragment pTH (1-34).

These peptides are capable of stimulating lipolysis, both in vitro and in vivo, through the activation of the membrane adenylate cyclase (for example: Tanigushi A. et al. J. Lip. Res. (1987) 28 .490-496). Unfortunately, the smaller of the two peptides mentioned above, pTH (1-

34), has a sequence of 34 amino acids (H-Ser-Nal-Ser-Glu-Ile-Gln-Leu-

Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Nal-Glu-Trp-Leu-Arg- Lys-Lys-Leu-Gln-Asp-Val-His- Asn-Phe-OH), which makes its synthesis on an industrial scale very difficult, and therefore its use incompatible with the economic requirements of the targeted cosmetic market.

25 Recently, a patent described the use of fragments of this pTH (1-34), Ν-acylated or not, composed of 3 to 10 amino acids, whose sequence started with Ser-Val, for their slimming effects by the topical (FR 98 09193). In view of all of the scientific literature (for example: Taniguchi A. et al. (1987) J. of Lipid Res. 28: 490-494), the presence of the two Ν-terminal amino acids

30 (Ser-Nal) then seemed to be compulsory. The object of this patent is the double discovery that the lipolytic activity, both in vivo and in vitro, was carried by peptide fragments, derived from pTH:

- even if the N-peptide side does not contain the classic Ser-Val sequence,

- even if the peptide only consists of 3 to 11 amino acids. These peptides therefore correspond to fragments which decline all the possibilities of length and cleavage of pTH (9-19).

The hybrid peptides, obtained by grafting the Ser-Nal dipeptide on the Ν-terminal side in the sequences described above, did not demonstrate lipolytic activity greater than that of the initial sequences. Surprisingly, the in vitro activity is found in vivo after topical application, and therefore in an approach relating to cosmetics. Due to their shorter lengths, the industrial synthesis of these peptides becomes economically realistic, and due to the high lipolytic activity of the sequences described here, it is entirely possible to use these peptides in all cosmetic and dermopharmaceutical compositions.

The peptide, minimal sequence, consists of Gly-Lys-His when the undecapeptide, longest sequence, corresponds to His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-

Glue.

Finally, as in patent FR 98 09193, in order to make all these peptides even more active topically, it is advantageous to make them lipophilic by grafting a fatty acid of more or less long chain (mirystyl, palmityl, stearyl, lipoyl ...) on the Ν-terminal amine and / or to esterify the carboxyl group of the peptide. Are therefore concerned by this patent, the various fragments of the peptide pTH (9-19), characterized in that they contain the following sequence of general structure:

where R = H, or an alkyl chain, linear or branched, saturated or unsaturated, hydroxylated or sulfurized or not, containing 1 to 24 carbon atoms, preferably 12 to 18 carbon atoms, and R ₂ = OH or OR ₃ or NR R ₅ with R ₃ = an alkyl chain from C 1 to C ₂₄ , preferably either C 1 to C ₃ or C ₁₄ to C ₁₈ and with R-jR ₅ being independently each other = H or an alkyl chain of 1 to 12 carbon atoms, preferably 1 to 3 carbon atoms, and AA is all or part of the following peptide sequence His-Asn-Leu-Gly-Lys - His-Leu-Asn-Ser-Met-Glu (pTH (9-19)), preferably Gly-Lys-His-Leu-Asn (pTH (12-16)) or even Gly-Lys-His (pTH (12 -14)).

The peptides which respond to the sequences described above have a real and significant lipolytic activity by the topical route which is usable in cosmetics and dermopharmacy.

The peptides, which are the subject of the patent, can be obtained either by conventional chemical synthesis (in solid phase or in homogeneous liquid phase), or by enzymatic synthesis (Kullman et al, J. Biol. Chem. 1980, 255, 8234) from constituent amino acids or their derivatives.

The peptides can also be obtained by fermentation of a strain of bacteria modified or not by genetic engineering, to produce the desired sequences or their different fragments.

Finally, the peptides can be obtained by extraction of proteins of animal or vegetable origin, preferably vegetable, followed by a controlled hydrolysis which releases the peptide fragments in question, with the stipulation that the released fragments correspond to the peptide sequences pTH (9- n) with n between 10 and 19 inclusive. Many proteins found in plants are likely to contain these sequences within their structure. The controlled hydrolysis makes it possible to release these peptide fragments.

To carry out the invention, it is possible, but not necessary, to extract either the proteins concerned first and then hydrolyze them, or to carry out the hydrolysis first on a crude extract and to purify the peptide fragments then. It is also possible to use the hydrolyzate without extracting the peptide fragments in question therefrom, making sure however to have stopped the enzymatic hydrolysis reaction in time and to measure the presence of the peptides in question by appropriate analytical means (tracing by radioactivity, immunofluorescence or immunoprecipitation with specific antibodies, etc.). Other simpler or more complex processes leading to cheaper or purer products are easily conceivable by those skilled in the art knowing the art of extracting and purifying proteins and peptides.

By way of example illustrating the invention, a few cosmetic formulas which are representative but not limiting of the invention are cited:

Example 1: Slimming gel

Carbopol 1342R 0.3

Propylene glycol 2.0

Glycerin 1.0

White petrolatum 1.5

Cylomethicone 6.0

Cetyl alcohol 0.5

LubrajelR MS 10 triethanolamine 0.3

Gly-Lys-His 0.01

Water, preservatives, perfume qs 100 g.

Example 2: Slimming cream

Steareth-21 2.4

Steareth-2 2.6

PPG-15 stearyl ether 8.0

Beeswax 0.5

Stearoxy dimethicone 3.0

Propylene glycol 3.0

CarbopolR 941 0.25

0.25 triethanolamine

N-Palmitoyl-Gly-Lys-His-Leu-Asn 0.01

Caffeine 1.0

Water, preservatives, perfumes qs 100 g

-ample 3: Alcoholic lotion

Ethanol 5.0

Propylene glycol 2.0 Dimethicone copolyol 0.5

PPG- 1 -PEG-9 lauryl glycol ether 0.6

N-Palmitoyl-His-Asn-Leu-Gly-Lys-His 0.001 water, preservatives, perfume qs 100 g The activity of the peptides will be demonstrated by the following two examples:

Example 4: Lipolytic activity in vitro In this example, 5 peptide sequences were tested:

Peptide 1: His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu, i.e. the longest sequence described in this patent, Peptide 2: His-Asn-Leu-Gly-Lys-His -Leu-Asn,

Peptide 3: Gly-Lys-His-Leu-Asn- S er-Met-Glu, Peptide 4: Gly-Lys-His-Leu-Asn,

Peptide 5: Gly-Lys-His, the shortest sequence in this patent. Human adipocytes (obtained from plastic surgery waste) are suspended in a survival medium.

One of the 4 preceding peptides is then added at different concentrations and, after 2 hours of incubation at 37 ° C., the amount of glycerol and fatty acids released in the external medium is measured.

Under the same conditions, the basic lipolytic metabolism and a series of positive controls are carried out, respectively in the absence of the peptide tested or in the presence of different concentrations of pTH (1-34) which is then considered as a reference product.

The following table shows the amounts of glycerol released (nmol / 2.5.10 ⁵ cells / 2hr) as well as the percentage increase (compared to basal level) observed with the peptides tested under these conditions under a final concentration of 1.10 ^"6 M.

Basai pTH (1-34) Peptide 1 Peptide 2 Peptide 3 Peptide 4 Peptide 5 Olycerol 17.5 ± 1.1 30.5 ± 1.1 27.7 ± 0.8 24.3 ± 0.8 25.6 ± 0.9 23.7 ± 0.5 20.3 ± 0.6

A% - + 75.1 + 58.2 + 39.2 + 46.2 + 35.4 + 16.8

Under the same experimental conditions, it is also possible to follow the variation in the concentration of cAMP (cyclic adenosine monophosphate) in the cell medium. When lower concentrations of peptides were tested, a concentration dependent effect was demonstrated on the release of glycerol, which clearly demonstrates the specificity of the biochemical and physiological mechanism involved. Example n ° 5: Lipolytic activity in vivo An in vivo test, carried out on 15 women aged 35 to 62 years for four weeks, consisted in monitoring the evolution of two parameters, the perimeter of the thighs measured using one centimeter and the thickness of the adipose layer determined using the ultrasound technique. The results given below therefore relate to the differences observed between the values obtained for these two parameters between time 0 and at the end of the test, ie 4 weeks later.

The gel described in Example No. 1 was used, except that the peptide was absent from the placebo gel.

After 4 weeks of twice-daily treatment with gels (a placebo gel and a gel containing the Gly-Lys-His-Leu-Asn peptide, there is a favorable change in the perimeter and thickness of the fatty layer on the thighs treated with the preparation containing the lipolytic peptide: -12% and -14% respectively, while the variations in the values of these two parameters on the thighs treated with placebo are not significant. The lipolytic peptides are used alone or in combination with one another , in a finished cosmetic or dermopharmaceutical product, in any galenic form: O / W and W / O emulsions, milks, lotions, gelling and viscous polymers, surfactants and emulsifiers, ointments, hair lotions, shampoos, soaps, powders, sticks and pencils , sprays, body oils. It is possible to use these peptides in the form of a solution, dispersion, emulsion, or encapsulated in vectors such as macro-, micro- or nan ocapsules, liposomes or chylomicrons, or included in macro-, micro- or nanoparticles, or in micro-sponges, or adsorbed on powdery organic polymers, talcs, bentonites and other mineral supports. The concentration of use of these peptides can vary between 0.000001 and 1% (w / w), preferably between 0.0001 and 0.1% in the finished product. These peptides can be combined in cosmetic compositions with any other ingredient usually used in cosmetics: extraction and / or synthetic lipids, gelling and viscosifying polymers, surfactants and emulsifiers, water-soluble or liposoluble active principles, extracts from other plants, tissue extracts, marine extracts, etc.

These peptides are obtained by extraction of plant proteins, followed by enzymatic hydrolysis so as to generate peptide fragments of average size less than 1500 daltons, part of the released fragments having to contain at least one of the sequences corresponding to pTH (9-n ) with n between 10 and 19 inclusive. The combination with other lipolysis stimulating agents such as caffeine, theophylline, xanthine derivatives in general is particularly advantageous for carrying out the invention.

These peptides are used in cosmetic or dermopharmaceutical compositions for cosmetic applications with lipolytic activity for skin care, particularly the slimming treatment of overweight thighs and hips, in the treatment of cellulite and in skin tightening.

These peptides or the cosmetic or dermopharmaceutical compositions containing them are used for the preparation of a medicament for skin care, particularly for the slimming treatment of overweight thighs and hips, in the treatment of cellulite and in firming skin.

Claims

claims

1. Peptide fragments of pTH (l-34) with the following structure:

Rι-NH-AA-R ₂ with

R _\ = H, or an alkyl chain, linear or branched, saturated or unsaturated, hydroxylated or sulfurized or not, containing 1 to 24 carbon atoms, preferably 12 to 18 carbon atoms,

R = OH or OR ₃ or NR ₄ R ₅ with R ₃ = an alkyl chain of Ci to C ₂₄ , preferably either Ci to C ₃ , or C ₁₄ to ₈ and with R.jR ₅ being independently one of the other = H or an alkyl chain of 1 to 12 carbon atoms, preferably of 1 to 3 carbon atoms,

AA is, all or part of variable length of the following peptide sequence His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu (pTH (9-19)), of which preferably Gly-Lys- His-Leu-Asn (pTH (12-16)) or Gly-Lys-His (pTH (12-14)), but excluding the sequences (pTH (9-18)), (pTH (10-18 )), (pTH (11-18)), (pTH (12-18)), (pTH (13-18)), (pTH (14-18)), (pTH (9-17)),

(pTH (9-16)), (pTH (9-15)), (pTH (9-14)), (pTH (9-13)).

2. Peptides according to claim 1 obtained by chemical synthesis, by enzymatic route, by fermentation or by extraction of proteins of vegetable origin.

3. Peptides according to 1 to 2 obtained by conventional peptide synthesis in homogeneous or heterogeneous phase or by enzymatic synthesis from the constituent amino acids.

4. Peptides according to claims 1 to 3 obtained by extraction of plant proteins, followed by enzymatic hydrolysis so as to generate peptide fragments of average size less than 1500 daltons, part of the released fragments having to contain at least one of the corresponding sequence pTH (9-n) with n between 10 and 19 inclusive.

5. Peptides according to claims 1 to 4, characterized in that their lipophilicity is increased by grafting a fatty acid of more or less long chain (mirystyl, palmityl, stearyl, lipoyl, etc.) onto the N-terminal amine and / or to esterify the carboxyl group of the peptide.

6. Use of the peptides according to claims 1 to 5, alone or in combination with one another, in a finished cosmetic or dermopharmaceutical product, at concentrations varying between 0.000001 and 1% (w / w), preferably between 0.0001 and 0.1%.

7. Use of the peptides according to claims 1 to 5 and claim 6, alone or in combination with one another, in a finished cosmetic or dermopharmaceutical product, in the form of a solution, dispersion, emulsion, or encapsulated in vectors such as macro-, micro- or nanocapsules, liposomes or chylomicrons, or included in macro-, micro- or nanoparticles, or in micro-sponges, or adsorbed on powdery organic polymers, talcs, bentonites and other mineral supports.

8. Use of the peptides, according to claims 1 to 5 and 6 to 7, alone or in combination with one another, in a finished cosmetic or dermopharmaceutical product, in any galenical form: O / W and W / O emulsions, milks, lotions, gelling and viscosifying polymers, surfactants and emulsifiers, ointments, hair lotions, shampoos, soaps, powders, sticks and pencils, sprays, body oils.

9. Use of the peptides according to claims 1 to 5 and 6 to 8, alone or in combination with one another, in a finished cosmetic or dermopharmaceutical product, with any other ingredient usually used: extraction and or synthetic lipids, gelling polymers and viscosants, surfactants and emulsifiers, water- or fat-soluble active ingredients, plant extracts, tissue extracts, marine extracts, caffeine, theophylline, xanthine derivatives and other lipolytic agents.

10. Cosmetic or dermopharmaceutical compositions containing the peptides according to claims 1 to 5 and 6 to 9 used in cosmetic applications with lipolytic activity for skin care, particularly the slimming treatment of overweight thighs and hips, in the treatment cellulite and in skin tightening.

11. Use of the peptides according to claims 1 to 5, or of a cosmetic or dermopharmaceutical composition containing the peptides according to claims 6 to 9, for the preparation of a medicament for skin care, particularly for the slimming treatment of overweight thighs and hips, in the treatment of cellulite and in skin tightening.