FR2781231A1 - New parathyroid hormone fragment peptides, used as lipolysis stimulants in topically applied cosmetic compositions for slimming treatment of excessive weight in hips and thighs - Google Patents

Abstract

Parathyroid hormone (pTH) (1-34) fragment peptides (I) are new. pTH (1-34) fragment peptides of formula R1-NH-AAn-Q (I) are new. R1 = optionally unsaturated, optionally hydroxylated or sulfurized 1-24C (preferably 12-18C) alkyl; Q = OR2 or NR3R4; R2 = H or 3-24C (preferably 14-18C) alkyl; R3, R4 = H or 1-12C (preferably 1-3C) alkyl; AA = all or part of the peptide sequence Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn (pTH (1-10)); n = 3-10. An Independent claim is also included for a cosmetic or skin pharmaceutical composition comprising (I), used for the treatment of cellulite and for skin firming, in the hips and thighs.

Description

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 xanthine derivatives; complex mixtures such as certain plant extracts, etc.) are offered and used.

  The market nevertheless demands new products and even more active products.

  Adrenergic substances (adrenaline and the like) are well known for their impressive ability to stimulate lipolysis in adipocytes but, their

  use is strictly prohibited in cosmetics and dermopharmacy.

  1 0 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-Val-Ser-Glu-IleGln-

  2 o Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-ValGlu-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.

  The object of this patent is the discovery that shorter peptide sequences. and therefore more realistic as to the possibility of carrying out industrial synthesis thereof, as well as certain chemically modified analogues are also active in the in vitro and in vivo stimulation of lipolysis. Contrary to all expectations, this activity is found in vivo after topical application, and therefore

  in a cosmetic approach.

  Are therefore concerned by this present patent, the various fragments of the peptide pTH-

  (1-34), characterized in that they contain the following sequence of general form: R '-NH-AAn-O-R2 o R' = H, or an alkyl chain, linear or branched, saturated or unsaturated, hydroxylated or sulfur-containing or not, containing 1 to 24 carbon atoms, preferably 12 to 18 carbon atoms, and R2 = H, or an alkyl chain from C1 to C24, preferably either CI to C3, or C14 to C1 8 or O-R2 = NR3R4 with R3 and R4 being independently of each other z o0 = H or an alkyl chain of 1 to 12 carbon atoms, preferably of 1 to 3 carbon atoms,

  and AA is all or part of the following peptide sequence: Ser-Val-SerGlu-Ile-

  Gln-Leu-Met-His-Asn (pTH (1-10)), preferably Ser-Val-Ser-Glu-IleGln

  (pTH (1-6)), and n between 3 and 10 inclusive, preferably between 3 and 6.

  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.

  Finally, 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 N-terminal amine

  and / or to esterify the carboxyl group of the peptide.

  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 acids

  2 5 constituent amines 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 sequences

  sought 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 ( 1-n) with n between 3 and 10 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 to hydrolyze them subsequently, 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

  the profession of extraction and purification of 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 No. 1: Carbopol l342R slimming gel 0.3 Propylene glycol 2, 0 Glycerin 1.0 White petrolatum 1.5 Cylomethicone 6.0 Sipol C16C18S3 0.5 LubrajelR MS 10 triethanolamine 0.3 N- Acetyl-Ser-Val-Ser-Glu-Ile-Gln-OH 0.001 N-Palmitoyl-Ser- Val-Ser-Glu-Ile-Gln-OH 0.00005

  3 o Water, preservatives, perfume qs 100 g.

  Example 2: Slimming cream BrijR 721 2.4 BrijR 72 2.6 ArlamolR E 8.0 Beeswax 0.5 AbilR ZP 2434 3.0 Propylene glycol 3.0 CarbopolR 941 0.25 Triethanolamine 0.25 N-Lipoyl-Ser-Val-Ser-Glu-Ile-Gln-OH 0.00001 Caffeine 1.0 Water, preservatives, perfumes qs 100 g Example 3: Alcoholic lotion Ethanol 5.0 s Propylene glycol 2.0 AbilR B8851 0.5 EumulginR L 0.6 H2N-Ser-Val-Ser-Glu-Ile-Gln-OMe 0.0002 water, preservatives, perfume qs 100 g The activity of the peptides will be demonstrated by the following two examples: Example No. 4: Lipolytic activity in vitro Human adipocytes (obtained from pieces of skin removed during plastic surgery) are suspended in a survival medium . Different concentrations of H-Ser-Val-Ser-Glu-Ile-Gln-OH (pTH (1-6)) are then added and, after 2 hours of incubation at 37 ° C., the amount of glycerol and of acids

  fat released into the outside environment.

  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.

3 o as a reference product.

  Under these conditions, the glycerol releases observed during five different tests are as follows: Basal 17.0 + 1.1 nmol / 2.5.105 cell / 2hr pTH (1-34) at 10'7M / 1 21.8 + 0.7 nmol / 2.5.105 cell / 2hr, pTH (1-6) at 10-6 M / 1 19.9 + 0.9 nmol / 2.5.105 cell / 2hr, which corresponds according to the peptide tested, increased release

  glycerol basal of 28% and 17% respectively.

  In this same model, we tested a number of peptides derived from the base sequence. For example: - to increase the lifetime of the peptide (with respect to proteolytic enzymes) we substituted glutamic acid in position 4 with D-glutamic acid and / or methionone in position 8 with norleucine, - to increase the affinity of the peptide for the skin, we attached an aliphatic chain (going from C1 to C24) either at the N-terminal end (bond

  amide), or at the C-terminal end (ester bond) of the peptide.

  In the presence of 1 * 10-6 M / l of product, the increases in basal glycerol release observed are as follows: pTH (1-10): + 18% pTH (1-8): + 10% 2 0 D -Glu4-pTH (1-6): + 17% Nle8-pTH (1-10): + 11% Pal-pTH (1-10): + 21% We therefore see that it is possible to modulate the activity sequence lipolytic

  main as needed (solubility, duration of action, epidermal affinity).

  Other variants can be envisaged by those skilled in the art while remaining within

the scope of the invention.

  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. In fact, the activation of lipolysis in adipocytes often, if not always, involves the stimulation of the activity of adenylate cyclase as a result of which the high concentration of cAMP leads to prolonged activation of triglycerides lipases. The peptides studied have various stimulating adenylate cyclase activities which are not linearly linked to lipolytic activity, which would indicate that different mechanisms for activating lipolysis intervene depending on the peptide. Some peptides are highly lipolytic without significantly stimulating adenylate cyclase, others have both activities to a high degree. The joint use of certain peptides which are the subject of this patent application

  can be particularly advantageous.

  Example No. 5: Lipolytic Activity in Vivo An in vivo test, carried out on 15 women aged 35 to 62 for four weeks, consisted in monitoring the evolution of two parameters, the perimeter of the thighs measured using a centimeter and the thickness of the adipose layer determined using the ultrasound technique. The results given below relate to the differences observed between the values obtained for these two

  parameters between time 0 and at the end of the test, 4 weeks later.

  The gel described in example n 1 was used, except that the two peptides were

absent from the placebo gel.

  After 4 weeks of twice daily treatment with gels (a placebo gel and a gel

  containing the two peptides N-Acetyl-Ser-Val-Ser-Glu-Ile-Gln-OH and NPalmitoyl-

  Ser-Val-Ser-Glu-Ile-Gln-OH respectively at 10-5 and 10-6 M / l), there is a favorable change in the perimeter and the thickness of the fatty layer on the thighs treated for the preparation containing lipolytic peptides: -13% and -15% respectively, while the decrease in these values on the thighs treated with

placebo is not significant.

  Peptides of lipolytic nature 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 viscosant polymers, surfactants and emulsifiers, ointments , hair lotions, shampoos, soaps,

  powder, 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 nanocapsules, liposomes or chylomicrons, or included in macro-, micro- or nanoparticles, or in micro-sponges, or adsorbed on polymers

  powdery organic, talcs, bentonites and other mineral supports.

  The concentration of use of these peptides can vary between 0.00001 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- or fat-soluble active principles, extracts from other plants, extracts tissues, 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 between 300 and 1500 daltons, part of the released fragments must contain at least one of the sequences corresponding to pTH (11 -n) with n between 3 and 10 inclusive. The combination with other lipolysis stimulating agents such as caffeine, theophylline, xanthine derivatives in general is particularly advantageous

to realize 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 the

skin firming.

  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 skin tightening.

Claims (11)

Claims   1. Peptide fragments of pTH (1-34) with the following structure: R'-NH-AAn-O-R2 o R '= H or a linear or branched alkyl chain, saturated or unsaturated, hydroxylated or sulfurized or not, containing 1 with 24 carbon atoms, preferably 12 to 18 carbon atoms, - R2 = H, or an alkyl chain from C1 to C24, preferably either CI to C3, or C14 to C18 or O-R2 = NR3R4 with R3 and R4 being independently from each other = H or an alkyl chain of 1 to 12 carbon atoms, o preferably of 1 to 3 carbon atoms,   - AA is all or part of the following peptide sequence: Ser-ValSer-Glu-   Ile-Gln-Leu-Met-His-Asn (pTH (-10)), preferably Ser-Val-Ser-Glu-   Ile-Gln (pTH (1-6)), and n between 3 and 10 inclusive, preferably between 3 and 6.   2. Peptides according to claim 1 obtained by chemical synthesis, by   enzymatic, by fermentation or by extraction of proteins of vegetable origin.   3. Peptides according to 1 to 2 obtained by conventional peptide synthesis in homogeneous phase   or heterogeneous or by enzymatic synthesis from the constituent amino acids.   4. Peptides according to claims 1 to 3 obtained by extraction of proteins from   plants, followed by enzymatic hydrolysis so as to generate peptide fragments of average size between 300 and 1500 daltons, part of the fragments released must contain at least one of the sequences corresponding to   pTH (l -n) with n between 3 and 10 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 ...) on the N-terminal amine and / or to esterify the group peptide carboxyl.   6. Use of the peptides according to claims 1 to 4 or according to claim   , alone or in combination with each other, in a finished cosmetic or dermopharmaceutical product, at concentrations varying between 0.00001 and 1%   (w / w), preferably between 0.0001 and 0.1%.   7. Use of the peptides according to claims 1 to 4 and 6 or according to   claims 5 to 6, alone or in combination with each other, in a product   finished cosmetic or dermopharmaceutical, 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 4 and 6 to 7 or according to   claims 5 to 7, alone or in combination with each other, in a product   l0 finished cosmetic or dermopharmaceutical, in all dosage forms: O / W and W / O emulsions, milks, lotions, gelling and viscosifying polymers, surfactants and emulsifiers, ointments, hair lotions, shampoos, soaps, powder and sticks   pencils, sprays, body oils.   9. Use of the peptides according to claims 1 to 4 and 6 to 8 or according to   claims 5 to 8 alone or in combination with each other in a product   cosmetic or dermopharmaceutical finished, with any other ingredient usually used: extraction and or synthetic lipids, gelling and viscosifying polymers, surfactants and emulsifiers, water or liposoluble 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 5 to 8 used in cosmetic applications to   lipolytic activity for skin care, particularly the slimming treatment of overweight thighs and hips, in the   2 5 treatment of cellulite and in skin tightening.   11. Use of the peptides according to claims 1 to 4 or of a composition   cosmetic or dermopharmaceutical containing the peptides according to the   claims 5 to 8 for the preparation of a medicament for the care of   skin, particularly for the slimming treatment of overweight thighs and hips, in the treatment of cellulite and in the skin firming.