Pro-pigmenting peptides

Abstract

Provided is at least one peptide of formula (I) and its use, where formula (I) is as follows: X-(Xaa.sub.1).sub.n-Pro*-(Xaa.sub.2).sub.m-Y (I). In formula (I), n=0 and m=1. At the N terminal end of the peptide, X is selected from H, —CO—R.sub.1 and —SO.sub.2—R.sub.1. At the C terminal end of the peptide, Y is selected from OH, OR.sub.1, NH.sub.2, NHR.sub.1 or NR.sub.1R.sub.2, R.sub.1 and R.sub.2 being independently selected from an alkyle, aryle, aralkyle, alkylaryl, alkoxy and aryloxy group, that can be linear, branched, cyclic, poly-cyclic, non-saturated, hydroxylated, carbonylated, phosphorylated and/or sulphured, with the possibility to have in said group skeleton a O, S and/or N heteroatom. Pro* corresponds to a Proline, an analogue or derivative thereof.

Claims

1. A dipeptide of formula (I):
X-(Xaa1)n-Pro*-(Xaa2)m-Y  (I) Wherein n=0 and m=1; Xaa.sub.2 is Alanine (Ala, A); wherein at the N terminal end of the dipeptide of formula (I), X is selected from the group consisting of octanoyl (C.sub.8), decanoyl (C.sub.10), lauroyl (C.sub.12), myristoyl (C.sub.14), palmitoyl (C.sub.16), stearoyl (C.sub.18), biotinoyl, elaidoyl, oleoyl and lipoyl; wherein at the C terminal end of the dipeptide of formula (I), Y is selected from the group consisting of OH, OR1, NH2, NHR1 and NR1R2; wherein R1 and R2 are independent from each other and selected from the group consisting of an acyl, alkyl, aryl, aralkyl, alkylaryl, alkoxy and aryloxy group, wherein said acyl, alkyl, aryl, aralkyl, alkylaryl, alkoxy and aryloxy group can be linear, branched, cyclic, poly-cyclic, non-saturated, hydroxylated, carbonylated, phosphorylated and/or sulfured, and may include an O, S and/or N heteroatom; wherein Pro* is Proline; and wherein the dipeptide of formula (I) does not include the dipeptide wherein X=H and Y=OH.

2. The dipeptide of formula (I) according to claim 1, wherein X is selected from the group consisting of lauroyl (C.sub.12), myristoyl (C.sub.14) and palmitoyl (C.sub.16).

3. The dipeptide of formula (I) according to claim 2, wherein said dipeptide is Pal-PA-OH.

Description

DETAILED DESCRIPTION

(1) The following examples describe and illustrate some aspects of the invention. They should not be seen as limiting the disclosure, as they only provide useful information for understanding and implementation.

(2) A) Example of Synthesis of the Peptide According to the Invention, the Pal-PA-OH:

(3) The Pal-PA-OH peptide is prepared by peptidic synthesis. Proline is acylated on its amine function with an activated derivative of palmitic acid (palmitoyl chloride for example) in the presence of a base to obtain the palmitoyl-proline. The latter is then activated on its terminal acid function with a coupling agent (DCC (diclyclohexylcarbodiimide)/NHS (N-hydroxysuccinimide) or HBTU (2-1H-enzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)/HOBT (1-hydroxybenzotriazole)) to react with the alanine. After precipitation, washing and drying, the palmitoyl-prolyl-alanine product is obtained in a solid form.

(4) B) Preparation of a Composition According to the Invention Comprising the Pal-PA-OH Peptide of Example A).

(5) Starting Products:

(6) The Pal-PA-OH pur peptide, synthetized according the synthesis method explained above;

(7) Excipient: a mixture of fatty esters, selected to form an oil matrix, for example for forming a water-free composition for the formulation of subsequent cosmetic water-free end-compositions.

(8) Protocol:

(9) the Pal-PA-OH peptide is mixed with the excipient and placed under gentle stirring and heating until complete dissolution and clarity.

(10) C) In Vitro Evaluations

(11) The peptides of the invention have a number of remarkable effects presented below. Peptides prepared according to A) above and dissolved in a fatty excipient to form compositions according to Example B) were in vitro tested and demonstrated a pro-pigmenting activity and for some of them an additional pro-collagen activity, that are presented below. Comparative tests are also presented below.

(12) 1. Pro-Pigmenting Activity (Melanogenesis)

(13) a) Test on B16 Cells

(14) Mouse melanoma (B16 cells) were grown in their maintenance medium (DMEMc+10% FCS) in the presence of the products and positive references from the literature for 48 hours. At the end of the contact, the melanins were extracted from the cells and assayed by spectrophotometry. In parallel, the residual tyrosinase activity (dopa oxidase) of the cells was assayed by spectrophotometry in the presence of one of the substrates of the enzyme: the L-DOPA. An estimate of the viability of the cells was also performed at the end of culture using a protein assay by the BCA method.

(15) b) Test on Human Melanocytes

(16) Normal human melanocytes (HEMn cells) were seeded in their maintenance medium then brought into contact with the products and the positive controls (literature references) in a test medium suitable for the culture of melanocytes for 10 days. At the end of the contact, the melanin are extracted from cells and assayed by spectrophotometry. In parallel, the residual tyrosinase activity (dopa oxidase) of the cells was assayed by spectrophotometry in the presence of one of the substrates of the enzyme: the L-DOPA. An estimate of the viability of the cells was also performed at the end of culture using a protein assay by the BCA method.

(17) c) Results on Pro-Pigmentation

(18) TABLE-US-00004 Peptides On B16 cells On human melanocytes IBMX (isobutyl-methyl-xanthine) +++ +++ Pal-AQPR-OH (SEQ ID NO: 15) +++ Pal-AQPK-OH (SEQ ID NO: 16) +++ Pal-QPR-OH ++ Pal-YPR-OH ++++ ++ Pal-SPR-OH ++++ ++ Pal-LPR-OH ++++ +++ Myr-LPR-OH ++ Lau-LPR-OH +++ H-LPR-OH =0 Pal-APR-OH +++ Pal-PPR-OH +++ ++ Pal-QPK-NH.sub.2 ++ Pal-QPH-OH +++ Pal-QPM-OH +++ Pal-QPA-OH +++ ++ Pal-LPA-OH + ++ Myr-DPA-OH =0 Pal-SPA-OH ++ Pal-PPA-OH + Pal-DPA-OH =0 Myr-SPA-OH ++ Pal-LGA-OH =0 Pal-LPM-OH + Pal-IPM-OH + Pal-MPL-OH +++ Pal-PR-OH +++ + Myr-PR-OH + Pal-PM-OH +++ + Pal-PP-OH +++ Pal-PG-OH +++ Pal-PS-OH ++ Pal-PA-OH +++ H-PA-OH =0 Pal-GA-OH =0 Pal-AP-OH ++ Pal-GP-OH +

(19) The results show a more or less pro-pigmenting activity for a representative panel of peptides according to the invention.

(20) The Table Gives Also Comparative Results:

(21) The Pal-LGA-OH and Pal-GA-OH show no pro-pigmenting activity compared to Pal-LPA-OH and Pal-PA-OH showing that when Proline is substitute by a more flexible amino acid, here glycine, it annihilates the pro-pigmenting activity.

(22) It can also be seen that Myr-DPA-OH and Pal-DPA-OH, including an acid Xaa.sub.1 (Asp (D) aspartic acid) do not present either a pro-pigmenting activity.

(23) Furthermore, Pal-FPM-OH, with a nonpolar Xaa.sub.1 (Phenylalanine Phe (F)) does not present either a pro-pigmenting activity.

(24) Finally, H-LPR-OH and H-PA-OH have no pro-pigmenting activity unlike Pal-LPR and Pal-PA-OH respectively, which shows the importance of having peptides with a substitution at X or Y.

(25) 2. Pro-Pigmenting and Pro-Collagen Dual Activity

(26) Normal human fibroblasts are seeded and placed in contact with the peptides to test for 6 days in DMEMc+5% FCS. After the contact, the layers are fixed and collagen 1 fibers are visualized by immunostaining with a specific antibody. Quantification is then performed by image analysis. The positive control is TGF β 10.sup.−6%.

(27) TABLE-US-00005 Melanogenesis Melanogenesis on human Peptide on B16 cells melanocytes Coll 1 Pal-SPR-OH ++++ ++ + Pal-PPR-OH +++ + Pal-QPA-OH +++ ++ + Pal-LPA-OH + ++ +++ Myr-SPA-OH ++ ++ Pal-PM-OH +++ + ++ Pal-PA-OH +++ ++

(28) 3. Activity on Keratinocytes

(29) Human keratinocytes are brought to just confluence in KSFMc environment. The contact with the actives and therefore their evaluation is then done in KSFMc medium alone or with calcium (0.8 mm)*. Visual evaluation of differentiation takes place after 2, 4/5 and 7 days of contact. *Calcium differentiation allows the creation of link structure between the cells which leads to a better attachment of the basal layers.

(30) TABLE-US-00006 Peptide Keratinocyte differenciation Pal-KT - positive control +++ at 2.5 ppm Pal-LPR + at 10 ppm Pal-SPR + at 10 ppm Pal-PA + at 2 ppm Pal-LPA ++ at 3 ppm

(31) D) Galenics

(32) Active Ingredient According to the Invention:

(33) Formulas comprising a peptide of the invention either is in a hydrophobic excipient as in the previous examples or in a hydrophilic excipient.

(34) Different formulations are described below. Additional cosmetic active ingredients, in support and/or in complement of the activity of the active ingredient according to the invention if necessary can be added to the appropriate phase according to their hydrophobic or hydrophilic nature. These ingredients can be of any class according to their(s) function(s), place of application (body, face, neck, chest, hands, hair, eyelashes, eyebrows, body hair, etc.), final desired effect and target consumer, for example anti-aging, anti-wrinkle, moisturizing, anti-wrinkle, firming, anti-glycation, slimming, soothing, myo-relaxing, anti-redness, anti-stretch marks, etc. They are mentioned above in the text.

(35) 1) Cream Form

(36) TABLE-US-00007 Ingredient (INCI name) Weight % Phase A Sorbitan Stearate 3.00 Cyclopentasiloxane (and) Cyclohexasiloxane 2.00 Ethylhexyl Palmitate 3.00 Glyceryl Stearate (and) PEG-100 Stearate 3.00 Ethylhexyl Methoxycinnamate 1.00 Ethylhexyl Dimethyl PABA 1.00 Phase B Demineralised water Qsp 100 Ultrez 10 ™ (Carbomer) 0.40 Phase C Glycerin 5.00 Conservatives qs Phase D Peptide according to the invention in a fatty 2.00 excipient Phase E Potassium sorbate (Potassium Sorbate) 0.10 Phase F Sodium hydroxyde 30% (Sodium Hydroxide) 0.60 Demineralised water 6.00 Phase G Fragrance 0.10

(37) Protocol:

(38) Weigh phase A and heat to 75° C. in a water bath. Weigh phase B and let swell for 20 minutes. Melt phase C until dissolved and add to phase B. Heat phase (B+C) to 75° C. in a water bath. Pour phase A in phase (B+C) under stirring. Extemporaneously, add phase A to phase (A+B+C). Approximately at 45° C. add phase E and neutralize with phase F. Mix well. At 35° C., add phase G. Homogenize well. pH: 6.20.

(39) Examples of Ingredients that can be Added to this Formulation:

(40) CALMOSENSINE™: soothing active for sensitive skins marketed by Sederma (W1998/07744) comprising the Tyr-Arg lipo-dipeptide. It reduces discomfort feelings.

(41) NG Birch Sap™: skin toning and moisturizing marketed by Sederma.

(42) Shea Butter: having nourishing and protective properties for the treatment of skin stressed by the environment.

(43) 2) Oil Form, in Particular for a Spray

(44) TABLE-US-00008 Ingredient (INCI name) Weight % Phase A Crodamol GTCC ™ (Capric/Caprylic Triglyceride) Qsp 100 Ethylhexyl Dimethyl PABA 2.00 Ethylhexyl Methoxycinnamate 2.00 Crodamol OS ™ (Ethylhexyl Stearate) 20.00 Tocopherol Acetate 0.20 Phase B Peptide according to the invention in a fatty 2.00 excipient Phase C Fragrance 0.05

(45) Protocol:

(46) Weigh phase A and put under helix stirring. Add phase B to phase A under helix stirring. Mix well. Add phase C to phase (A+B).

(47) Examples of Ingredients that can be Added to this Formulation:

(48) REVIDRATE™: active marketed by Sederma (WO2011/086532) that in particular improves the cohesion of the epidermis and its hydration.

(49) RENOVAGE™: global anti-aging active ingredient marketed by Sederma (WO2006/120646).

(50) Gel Form

(51) TABLE-US-00009 Ingredient (INCI name) Weight % Phase A Demineralised water Qsp 100 Ultrez 10 ™ (Carbomer) 0.20 Phase B PEG 400 ™ 5.00 Conservatives qs Phase C Dimethicone 4.00 Pemulen TR2 ™ (Acrylates/C10-30 Alkyl Acrylate 0.20 Cross Polymer) Phase D Tween 20 ™ (Polysorbate 20) 1.00 Peptide according to the invention in a fatty 2.00 excipient Phase E Potassium sorbate (Potassium Sorbate) 0.10 Phase F Sodium hydroxyde 30% (Sodium Hydroxide) 0.60 Demineralised water 5.00 Phase G Fragrance 0.10

(52) Protocol:

(53) Disperse Ultrez 10 in water and let swell for 15 minutes. Heat phase B until dissolved and add to phase A. Weigh and mix phase C. Mix phase D and add it to phase C; mix well. Add phase (C+D) to the phase (A+B). Then add phase E. Let swell for 1 hour. Mix well. Neutralize with phase F. Finally, add phase G. pH: 6.10.

(54) Examples of Ingredients that can be Added to this Formulation:

(55) RESISTEM™: anti-aging marketed by Sederma (WO2012/104774), helping the skin to build its own anti-aging defense system, based on an extract obtained by cell culture of Globularia cordifolia plant.

(56) AQUALANCE™: osmoprotector moisturising active ingredient marketed by Sederma (WO2009/104118) comprising homarine and erythritol.

(57) LEGANCE™: anti-aging active marketed by Sederma (WO2013/105047), corresponding to a Zingiber zerumbet Smith extract obtained by C.sub.02 supercritical in a water-soluble excipient and titrated in zerumbone ingredient. It is a global anti-aging ingredient for legs. It improves their appearance and comfort by reducing water retention, improving microcirculation and refining adipose tissue.

(58) BODYFIT™: slimming/firming active ingredient comprising glaucine marketed by Sederma (WO2004/024695). BODYFIT™ reduces the appearance of cellulite and helps to improve drainage and water distribution in the tissues.

(59) PRODIZIA™: active ingredient marketed by Sederma (WO2013/046137) fighting the signs cutaneous fatigue caused by glycation and glycoxidation.

(60) JUVINITY™: active (WO2011/125039) marketed by Sederma reducing signs of aging on the face and neckline, smoothing wrinkles, densifying and restructuring the dermis.

(61) Compact Powder Form

(62) TABLE-US-00010 Ingredient (INCI name) Weight % Phase A Talc Qsp 100 Kaolin 2.00 Calcium Stearate 1.00 Mica 4.00 Silica 1.00 Bismuth Oxychloride 2.00 Potassium Sorbate qs Phenoxyethanol qs Phase B Unipure ™ Black LC 989 HLC [CI 77499 (and) 0.20 Hydrogenated Lecithin] Unipure ™ Red LC 381 HLC [CI 77491 (and) 0.60 Hydrogenated Lecithin] Unipure ™ Yellow LC 182 HLC [CI 77492 (and) 1.00 Hydrogenated Lecithin] Covapearl Star ™ Gold 2302 AS [CI 77891 (and) 0.50 CI 77491 (and) Synthetic Fluorphlogopite (and) Triethoxycaprylylsilane] Covapearl ™ Brown 838 HLC [CI 77491 (and) Mica 1.00 (and) Hydrogenated Lecithin) Covapearl ™ Dark Blue 637 [CI 77510 (&) 0.10 CI 77891 (&) Mica] Phase C Crodamol PTIS-LQ-(MV) ™ [Pentaerythrityl 4.00 Tetraisostearate] Peptide according to the invention in a fatty matrix 2.00 Phase D Fragrance 0.30

(63) Protocol:

(64) Weigh phase A and mix. Weigh phase B and pour into phase B. Pour (A+B) in the blender and mix. Add phase C to (A+B) in several times and mix each time. Add phase D. Check homogeneity at each step.

(65) Examples of ingredients that can be added to this formulation:

(66) VEGESOME MOIST 24™: ingredient marketed by Sederma designed for the formulation of moisturizing powder makeup; it is a powder consisting of hollow particles 25 microns (Lycopodium clavatum exins) loaded with an Imperata cylindrica extract having moisturizing properties.

(67) Other Cream Form

(68) TABLE-US-00011 Ingredient (INCI name) Weight % Phase A Arlacel 170 ™ (Glyceryl Stearate (and) PEG-100 5.50 Stearate) Abil Wax 2434 ™ (Stearoxy Dimethicone) 3.00 Acetulan ™ (Cetyl Acetate (and) Acetylated 1.50 Lanolin Alcohol) Crodacol C 90 ™ (Cetyl Alcohol) 1.50 Mineral Oil 3.00 Shea Butter 5.00 Unsaponifiable Shea 1.00 Parsol MCX ™ (Ethylhexyl Methoxicinnamate) 3.50 Phase B Demineralised water Qs 100 Phase C Carbopol 940 ™ (Carbomer) 0.20 Phase D Demineralised water 2.00 Triethanolamine 99% (Triethanolamine) 0.20 Phase E Propylene Glycol 0.10 Mixed Parabens Phase F Sodium hydroxyde 30% (Sodium Hydroxide) 5.00 Demineralised water qs Phase G Peptide according to the invention in an hydrophilic 2.00 excipient

(69) Protocol:

(70) Weigh phase A and heat at 75° C. in a water bath. Weigh phase B and let swell for 20 minutes. Melt phase C until dissolved and add it to phase B. Heat phase (B+C) at 75° C. in a water bath. Pour phase A in phase (B+C) under Staro stirring. Extemporaneously, add phase A to phase (A+B+C). At approximately 45° C. add phase E and neutralize with phase F. Mix well. At 35° C., add phase G. Homogenize well. pH: 6.20.

(71) Examples of Ingredients that can be Added to this Formulation:

(72) SUBLISKIN™: active ingredient marketed by Sederma (WO2010/067327) that moisturizes and smooths the skin while allowing it to resist to external aggressions.

(73) VENUCEANE™: active marketed by Sederma (WO2002/066668) comprising a Thermus thermophiles biotechnological extract, that prevents visible signs of photo-aging (spots, wrinkles, dryness . . . ), protects cell structures from damages caused by UV and strengthens skin integrity.

(74) MATRIXYL synthe'6™: peptide-based anti-wrinkle ingredient marketed by Sederma (WO2010/082175) which helps repair skin damage caused by aging.

(75) KOMBUCHKA™: active ingredient acting on complexion marketed by Sederma (WO2004/012650).

(76) INTENSLIM™: slimming active ingredient marketed by Sederma (WO2013/105048) which is a synergistic combination of extracts obtained by Globularia cordifolia plant cell culture, Zingiber zerumbet Smith titrated in zerumbone and vegetable caffeine obtained by supercritical CO.sub.2 extraction.

(77) Depigmenting Hair Lotion

(78) TABLE-US-00012 Ingredient (INCI name) Weight % Phase A Demineralised water Qsp 100 Ethanol 50.00 Phase B Methyl Paraben 0.20 Butylene Glycol 2.00 Phase C Tween 20 ™ [Polysorbate 20] 1.50 Fragrance 0.10 Phase D Peptide according to the invention 3.00

(79) Protocol:

(80) Weigh phase A. Weigh and melt phase B. Cool phase B. Mix phase B with phase A under propeller stirring. Mix phase C and add to phase (A+B). Add phase D to phase (A+B+C). pH: 6.70.

(81) Examples of Ingredients that can be Added to this Formulation:

(82) PROCAPIL™: anti-hair-loss active ingredient marketed by Sederma (WO 00/58347) that combines a vitamin matrikine (biotinyl-GHK), apigenin (a flavonoid extracted from citrus) and oleanolic acid (root extract from Loveyly hemsleya).