WO2007087738A1

WO2007087738A1 - Biologically active tripeptides and copper complexes and salts thereof

Info

Publication number: WO2007087738A1
Application number: PCT/CH2007/000050
Authority: WO
Inventors: Hugo Ziegler; Peter Wikstroem
Original assignee: Pentapharm Ag
Priority date: 2006-02-03
Filing date: 2007-02-01
Publication date: 2007-08-09

Abstract

Biologically active tripeptides of the general formula (I) in which R1 is hydrogen or C1-C16-alkylcarbonyl, R2 is 4-imidazolyl or -CH2NH2, R3 is hydrogen, C1-C16-alkyl or optionally substituted arylalkyl (C1-C4), -CH2-, -CH2CH2-, C (CH3)2, -NH-, or -CH(R4)-, where the [-CH(R4)-] group is D-configured; R4 is C1-C4-alkyl or benzyl and X is oxygen (-O-) or -NH-; 1:1 complexes of these tripeptides with Cu+2 [Cu(II)], as racemates or in their enantiomerically pure form; salts of the tripeptides of the formula (I) and salts of the 1:1 complexes of these tripeptides with Cu+2 [Cu(II)], with the proviso that, when R1 is hydrogen and R2 is 4-imidazolyl, neither is A -CH2-, -CH2-CH2- or -CH(R4)-, in which R4 is CH3, and X simultaneously oxygen; nor is A -CH2-, X simultaneously -NH- and R3 simultaneously hydrogen; and cosmetically dermatologically active compositions which comprise such compounds.

Description

Biologically active tripeptides, their copper complexes and salts

The present invention relates to biologically active tripeptides, their 1: 1 copper complexes and salts thereof.

introduction

It is known that after damage to body tissue and naturally occurring skin renewal by proteolysis, the tripeptide H-Gly-His-Lys-OH is released. The high affinity of this tripeptide for copper (II) ions allows it to dissolve copper (II) ions from carrier molecules such as albumin and form a copper (II) complex. This forms a 1: 1 complex (GHK-Cu), in which the copper. Ion is bound via the glycine nitrogen, the deprotonated nitrogen of the GIy-His bond and the imidazole nitrogen of the histidine, forming a quadratic pyramidal configuration with two water molecules (Loren Pickart, SpecChem Online 10/2002). This complex is a natural component of human plasma, saliva and urine.

The biological effect of GHK-Cu is manifold. GHK-Cu is a potent, selective activator for the skin in wound healing processes, removing scars or damaged tissue and forming and properly storing new tissue. GHK-Cu is involved in the construction of new skin tissue by enhancing angiogenesis and generally stimulates the synthesis of extracellular matrix (ECM) molecules, such as the production of collagen I and III, the accumulation of glycosaminoglycans and proteoglycans, e.g. Decorin (but not biglycan) as well as elastin. Studies showed that GHK-Cu also stimulated the chemotaxis of leukocytes.

Although GHK-Cu basically stimulates the synthesis of ECM molecules, in serum of free primary culture it was shown that GHK-Cu reduces the excretion of TGF-ß-1 from normal fibroblasts and especially in keloid-forming (scar-forming) fibroblasts. The improved healing of wounds or new formation of fresh Skin tissue by GHK-Cu is also based, inter alia, on a selective stimulation of MMP-9 and MMP-2 in an early or late phase of the wound healing process. This accelerates the breakdown of damaged protein and removes scars, and appropriately increases the synthesis of new collagen. Overall, there is improved wound healing with less scarring. Further effects are described in Cosmetics & Toiletries 118 (7), 24-28 (2003).

The positive effects of GHK-Cu in topical cosmetic application are i.a. an increase in skin thickness in the epidermis and dermis, improved skin hydration, significant smoothing of the skin through the stimulation of collagen synthesis, increased elasticity of the skin, marked improvement in skin contrast, increased production of collagen I, glycosaminoglycans and decorin Damaged skin (Nils Krüger et al., Cosmetic Medicine 2003 (1), 31-33; Alain Simeon et al., J. Invest Dermatol., 115, 962-968 (2000)).

The topical application of GHK-Cu, however, is associated with two serious disadvantages. The enzymatic stability of GHK-Cu to peptidases in the skin is very low. Within one minute, 95% GHK is degraded to glycine and HK. In addition, only between 0.1 and 0.3% of the applied amount can penetrate into the dermis. As a result, high concentrations (4%) of GHK-Cu have to be used for a still unsatisfactory effect (Loren Pickart, Published Studies on Copper-Peptide Induced Tissue Regeneration).

The object of the present invention was to find GHK-Cu analogous active ingredients whose stability is higher and / or their skin penetration rate significantly improved. It has now been found that, surprisingly, with selected artificial peptides, as they are defined herein below, stable 1: 1 copper complexes can be obtained and that the biological activity of these peptides not only remains intact but can even be improved in some cases. Description of the invention

The present invention relates to biologically active tripeptides of the general formula (I):

wherein

R ^{1 is} hydrogen or C ¹ -C 18 -alkylcarbonyl,

R ^{2 is} 4-imidazolyl or -CH ₂ NH ₂ ,

R ^{3 is} hydrogen, C ₁ -C ₆ -alkyl or optionally substituted

Arylalkyl (C 1 -C ₄ ), A is -CH ₂ -, -CH ₂ CH ₂ -, C (CH ₃ J ₂ , -NH- or -CH (R ⁴ ) -, where the group [-CH (R ⁴ ) -] is D-configured; R ^{4 is} C ₁ -C ₄ -alkyl or benzyl and X is oxygen (-O-) or -NH-;

1: 1 complexes of these tripeptides with Cu ⁺² [Cu (II)], as racemates or in their enantiomerically pure form; Salts of the tripeptides of the formula (I) and salts of the 1: 1 complexes of these tripeptides with Cu ⁺² [Cu (II)], with the proviso that when R ^{1 is} hydrogen and R ^{2 is} 4-imidazolyl, neither simultaneously

A is -CH ₂ -, -CH ₂ -CH ₂ - or -CH (R ⁴ ) -, wherein R ^{4 is} CH ₃ , and X is

Mean oxygen; still at the same time

A is -CH ₂ -, X is -NH- and R ^{3 is} hydrogen.

In a specific embodiment, in formula IA, -CH ₂ -, -CH ₂ CH ₂ -, -NH- or -CH (R ⁴ ) -, wherein the group is [-CH (R ⁴ ) -] D- configured.

In a further specific embodiment, not simultaneously A is -CH ₂ - or -CH (R ⁴ ) -, wherein R ^{4 is} -CH ₂ -CH ₃ or -CH (CH ₃ J ₂ , R ^{2 is} 4-imidazolinyl and X is oxygen.

In a preferred embodiment, in formula IR ² -CH ₂ NH ₂ . The present invention also relates to the use of the compounds of the formula (I), their copper complexes and salts thereof, as cosmetic active ingredients.

The present invention also relates to compositions, in particular cosmetic preparations, which comprise at least one compound of the formula (I), their copper complexes and / or their salts, and to processes for the preparation of these preparations.

The present invention also relates to compositions, in particular cosmetic preparations, which contain at least one compound of the formula (I), their copper complexes and / or salts thereof and at least one further dermatologically cosmetic active ingredient.

Under the definition "tripeptides of the general formula (I) and 1: 1 complexes of these tripeptides with Cu ⁺² [Cu (II)], as racemates or in their enantiomerically pure form, as well as their salts", the compounds of the formula (I ), their 1: 1 copper (II) complexes, salts of these copper (II) complexes with acids, and salts, ie salts of the tripeptides of the formula (I) per se and salts of 1: 1 complexes of the tripeptides of the formula (I ., to understand) with Cu ⁺² [Cu (II)], in particular those salts with acids, which salts are formed by the basic amino groups of the compounds of formula (I) with acids, preferably the 1: 1 copper (II) Complexes and salts of these copper (II) complexes with acids.

The general terms used above are defined as follows. By "alkyl" as a group per se and as a structural element for alkyl-containing groups are meant both linear and branched saturated hydrocarbon radicals. Examples are methyl, ethyl, propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-undecanyl, n-dodecanyl, n-tridecanyl, n-hexadecanyl, n- Heptadecanyl, n-octadecanyl and n-nonadecanyl as unbranched radicals and as branched radicals: isopropyl, tert-butyl, isobutyl, sec-butyl and isoamyl. "Aryl" represents aromatic hydrocarbon radicals, such as phenyl and naphthyl; preferred is phenyl.

Examples of substituents of the optionally substituted aryl groups are, for example, halogen, C ₁ -C ₅ -Al] CyI, hydroxy, C ₁ -C ₆ -alkoxy, C ₁ -C ₆ -alkoxycarbonyl, -CN, amino (-NH ₂ ), Ci C ₆ alkylamino, di (C ₁ -C ₆ ) alkylamino, aminocarbonyl, C ₁ -C ₆ -alkylaminocarbonyl or di (C ₁ -C ₆ ) -alkylaminocarbonyl.

"Halogen" means fluorine, chlorine, bromine and iodine; preferred are fluorine and chlorine.

The group [-CH (R ⁴ ) -] (as one of the meanings of "A") in the compound of formula (I) is derived from the amino acid H ₂ NA-COOH, which is in the unnatural D configuration.

In formula (I), R ^{1 is} preferably hydrogen or CH ₃ C (O) -. R ³ is preferably hydrogen or benzyl. R ⁴ is preferably methyl.

The basic amino groups of the compounds of formula (I) and / or their copper complexes can turn with acids or multidentate _/ form homogeneous or mixed salts, for example with inorganic acids such as hydrogen chloride, hydrogen bromide, sulfuric acid or phosphoric acid; or with suitable carboxylic acids, for example aliphatic mono- or dicarboxylic acids, such as formic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, propionic acid, glycolic acid, succinic acid, fumaric acid, malonic acid, maleic acid, oxalic acid, phthalic acid, citric acid, lactic acid or tartaric acid; or with aromatic carboxylic acids, such as benzoic acid or salicylic acid; or with aromatic-aliphatic carboxylic acids, such as mandelic acid or cinnamic acid; or with heteroaromatic carboxylic acids, such as nicotinic acid; or with aliphatic or aromatic sulfonic acids, such as methanesulfonic acid or toluenesulfonic acid. Preference is given to dermatologically acceptable salts.

The individual compounds in the following Tables 1 and 2 illustrate the invention.

Table 2: Copper (II) complexes of the general formula I.

The general formula (I) includes all possible isomeric forms as well as their mixtures, for example racemic mixtures and mixtures of rotamers.

The copper-free compounds of general formula I can be prepared according to known methods (1994 general rules of M. Bodanszky "The Practice of Peptide Synthesis" Springer Verlag, 2 ^nd Edition). Accordingly, the P-amino acid is derivatized at the carboxy-terminal end, deprotected the α-amino protecting group (eg a Boc group) and the peptide gradually built up with the customary in peptide synthesis reagents until the desired sequence is fully established. Subsequently, the side-chain protective groups (eg Z or Boc functions) are cleaved off and the tripeptide derivatives are purified by chromatography and / or by recrystallization.

To prepare the copper complexes of general formula (I), the copper-free, purified peptide derivatives are dissolved in water or alcohol / water and reacted with the equimolar amount of a copper salt, e.g. Copper (II) chloride and copper (II) acetate or copper (II) hydroxide are added and the pH of the solution is adjusted, typically with sodium hydroxide (NaOH).

The compounds according to the invention can be used in concentrations which vary between 0.5 and 20,000 ppm (w / w), preferably between 1 and 5,000 ppm (w / w), in the final cosmetic product.

The compounds according to the invention may be in the form of a solution, a dispersion, an emulsion or encapsulated in carriers such as the macro-, micro- or nanocapsules, in liposomes or chylomicrons, or included in macro, micro or nanoparticles or in microsponges or absorbed on powdered organic polymers, talc, bentonite and other mineral carriers.

The compounds of this invention may be used in any galenic form: aqueous / oily and oily / aqueous emulsions, milk, lotions, ointments, gelling and viscous, stress active and emulsifying polymers, pomades, shampoos, soaps, gels, powders, sticks and sticks, sprays , Body oils, face masks, plasters.

The compounds according to the invention can be used with any other cosmetic ingredient commonly used: extraction lipids and / or synthetic lipids, gelling and viscous, active and emulsifying polymers, water or fat soluble active principles, plant extracts, tissue extracts, marine extracts, sunscreens, antioxidants, moisturizers and barrier agents. Skin revitalizing agents.

The compounds of the invention can be used with any other commonly used cosmetic skin care active ingredient. Exemplary of an additional skin care active ingredient are anti-wrinkle active ingredients / anti-atrophic agents: The compositions according to the invention may contain a safe and effective amount of one or more anti-wrinkle active ingredients or anti-atrophic ingredients. Exemplary anti-wrinkle / anti-atrophic agents suitable for use in the compositions of the invention include sulfur-containing D and L amino acids and their derivatives and salts, especially the N-acetyl derivatives, a preferred example of which is N-acetyl-L- cysteine is; Thiols, such as ethanethiol; Hydroxy acids (eg .alpha.-hydroxy acids such as lactic acid and glycolic acid or .beta.-hydroxy acids such as salicylic acid and salicylic acid derivatives such as the octanoyl derivatives), phytic acid, lipoic acid; Lysophosphatidic acid, skin peeling agents (eg, phenol and the like), vitamin B ₃ compounds, and retinoids, which improve the skin smoothing benefits of the present invention. a) Vitamin B3 compounds

The compositions of this invention may contain a safe and effective amount of a vitamin B3 compound. Vitamin B3 compounds are particularly useful for the regulation of skin condition, as described in copending U.S. Patent Application Serial No. 08 / 834,010, filed April 11, 1997 (corresponding to International Publication WO 97/39733 A1, published on May 30, 1997) October 30, 1997). Exemplary derivatives of said vitamin B ₃ compounds include nicotinic acid esters including non-vasodilating esters of nicotinic acid (eg, tocopheryl nicotinate), nicotinyl amino acids, nicotinyl alcohol esters of carboxylic acids, nicotinic acid N-oxide, and niacinamide N-oxide.

b) Retinoids

The compositions of the invention may also contain a retinoid. "Retinoid" as used herein includes all natural and / or synthetic analogs of vitamin A or retinol-like compounds that have biological activity of vitamin A in the skin, as well as the geometric isomers and stereoisomers of these compounds. The retinoid is preferably retinol, retinol esters (eg, C ₂ to C ₂₂ alkyl esters of retinol including retinyl palmitate, retinyl acetate, retinyl propionate), retinal and / or retinoic acid (including all-trans retinoic acid and / or 13-cis retinoic acid ), in particular retinoids other than retinoic acid. Other suitable retinoids are tocopheryl retinoate [tocopherol esters of retinoic acid (trans or ice)], adapters {6- [3- (1-adamantyl) -4-methoxyphenyl] -2-naphthoic acid} and tazarotene (ethyl-6- [2- (4 , 4-dimethylthiochroman-6-yl) ethynyl] nicotinate). Preferred retinoids are retinol, retinyl palmitate, retinyl acetate, retinyl propionate, retinal and combinations thereof. The compositions of this invention may contain a safe and effective amount of the retinoid such that the resulting composition is safe and effective for regulating the condition of horny tissue, preferably for regulating visible and / or tactile discontinuities of the skin, particularly for regulating signs of aging of the skin. more preferably for the regulation of visible and / or tactile Skin surface texture discontinuities associated with skin aging.

(c) hydroxy acids

The compositions of this invention may contain a safe and effective amount of a hydroxy acid. Preferred hydroxy acids for use in the compositions of this invention include salicylic acid and salicylic acid derivatives.

d) peptides

Additional peptides include, but are not limited to, di-, tri-, tetra-, penta- and hexapeptides. Derivatives thereof may be added to the compositions of this invention in safe and effective amounts. As used herein, "peptides" refers to both the naturally occurring peptides and the synthesized peptides, and also includes peptidomimetics and metal complexes of "peptides". The naturally occurring and commercially available compositions containing peptides are also useful herein.

Suitable dipeptides for use herein include carnosine (β-Ala-His). Suitable tripeptides for use herein include Gly-His-Lys, Arg-Lys-Arg, His-Gly-Gly. Preferred tripeptides and derivatives thereof include palmitoyl-Lys-Val-Lys, which as SYN ^® -COLL commercially available from Pentapharm, Switzerland is available, palmitoyl-Gly-His-Lys, which can be purchased as Biopeptide CL ™ (100 ppm palmitoyl-Gly He-Lys, commercially available from Sederma, France), peptide CK (Arg-Lys-Arg), peptide CK + (ac-Arg-Lys-Arg-NH ₂ ), and β-Ala-Pro-Dab-NH-benzyl, which is sold under the name SYN ^® -AKE of Pentapharm, Switzerland, one. Tetrapeptides suitable for use herein include Peptide E, Arg-Ser-Arg-Lys. Examples of pentapeptides are Matrixyl (palmitoyl-Lys-Thr-Thr-Lys-Ser), available from Sederma, France, and those described in WO 03/037933 (Pentapharm, Switzerland). A hexapeptide suitable for use is Argireline (Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2) manufactured by Lipotec, Spain. Experimental part

1. Preparation of the compounds

The following examples are intended to illustrate the invention without limiting it.

Used abbreviations:

NMR Magnetic nuclear spin resonance

MS mass spectrometry

Boc tert. butyloxycarbonyl

Z is benzyloxycarbonyl

Dab 1,4-diaminobutyric acid

EtOAc ethyl acetate

DCM dichloromethane

DMF dimethylformamide

ACN acetonitrile

TCTU O- (1H-6-chlorobenzotriazole-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate DIPEA diisopropylethylamine

NMM N-methylmo N-hydroxysuccinimide ester

TFA trifluoroacetic acid

GF / A fiberglass microfilter

LH20 size fraction chromatography

( ^R Amersham)

In the following exemplary embodiment 1, the preparation of compounds of the formula I according to the invention is described. The

Analysis of the eluates and products obtained according to the examples was carried out by proton NMR spectroscopy, HPLC electrospray MS or by elemental analysis. The connections could open

ZIC-HILIC columns (zwitterion chromatography hydrophobicity

Interaction Chromatography Column), resulting in meaningful HPLC chromatograms.

Column: SeQuant ZIC ™ -HILIC, 5um. 150x6.6mm)

Eluant A: A - 85% (v / v) ACN with 15% (v / v) 10 mM KH ₂ PO ₄ buffer, pH

7

B - 10 oMM KH ₂ PO ₄ buffer, pH 7. Linear gradient: from 10% B to 100% B in 20min.

The compounds can be by the methods described below to known methods (general rules of M. Bodanszky "The Practice of Peptide Synthesis" Springer Verlag, 2 ^nd Edition 1994) are manufactured. Accordingly, the P-amino acid is derivatized at the carboxy-terminal end, the α-amino-protecting group (eg a Boc group) is deprotected and the peptide is built up stepwise with the reagents customary in peptide synthesis until the desired sequence is completely established. Subsequently, the side chain protection groups (eg Z or Boc functions) are split off.

Example 1: Copper (II) - Glycyl-2,4-diaminobuturyl-lysine complex

Acetate dihydrate

Cu [H-Gly-Dab-Lys-OH] x HOAc x 2 (H ₂ O)

1.1) Boc-Dab (Z) -Lys (Z) -OBzI

26.8 g of Boc-Dab (Z) -OH were dissolved in 240 ml of ACN and 60 ml of DMF and to the solution were added 27 g of TCTU and 26 ml of DIPEA. After stirring for 5 minutes at RT, a solution consisting of 34 g of H-Lys (Z) -OBzI • HCl and 8.4 ml of NiMM in 100 ml of DMF was added. After stirring at RT overnight, the solvent was distilled off, the residue taken up in EtOAc and washed with 10% Na ₂ CO ₃ , 10% citric acid and sat. NaCl solution washed. After drying over Na ₂ SO ₄ and distilling off the solvent, the crude product was purified by chromatography over LH20. After distilling off the solvent and drying in a vacuum oven at 4O ⁰ C overnight, 38.2 g of the desired compound were obtained. The theoretical mass of 706 was confirmed with a finding of 706.

1.2) H-Dab (Z) -Lys (Z) -OBzI x HCl

38.2 g of compound 1.1 were dissolved in 150 ml of dioxane and combined with 136 ml of 4M HCl in dioxane solution and stirred at RT for 3 h. After distilling off the solvent, 35 g of the desired compound were obtained. Its molecular weight could be confirmed with a mass of 606. 1.3) Boc-Gly-Dab (Z) -Lys (Z) -OBzI

22.1 g of Boc-Gly-OSu were dissolved in 150 ml of DMF. A solution consisting of 35 g of the above compound 1.2 and 6 ml of NMM in 300 ml of DMF was added thereto. After 1.5 days of stirring at RT, the solvent was distilled off, the residue taken up in EtOAc and washed with 3 X 10% Na ₂ CO ₃ , 10% citric acid and sat. Washed NaCl solution. After drying over Na ₂ SO ₄ and distilling off the solvent, the crude product was purified over LH20 in methanol. After distilling off the methanol and drying in a vacuum oven at 40 ⁰ C overnight, 31.4 g of amorphous product were obtained. Its molecular weight could be confirmed with a mass finding of 763.

1.4) H-Gly-Dab (Z) -Lys (Z) -OBzI x TFA

12.4 g of the above compound 1.3 were dissolved in 30 ml of DCM and admixed with 12 ml of TFA and stirred at RT for 1.5 h. After distilling off the solvent, the crude product was chromatographed on LH20. After drying in a vacuum oven at 4O ⁰ C overnight 12.4 g of product whose molecular weight was confirmed by a mass finding of the 663rd

1.5) H-Gly-dab-Lys-OH x 2HOAc

12.02 g of the above compound 1.4 were dissolved in 100 ml of acetic acid and the solution was treated with a suspension of 2.4 g of palladium (10% on carbon) in 20 ml of water. Hydrogen gas was bubbled to saturation and hydrogenated at atmospheric pressure until complete reaction (about 12 hours). The suspension was filtered through a GF / A filter and the solvent distilled off. The resulting crude product was dissolved in about 100 ml of deionized water, filtered and diluted with a further 400 ml of deionized water and salted in an ion exchange column (OAc ^" ) .The resulting amorphous crude product (6.45 g) was chromatographed by preparative HPLC after distilling off the solvent and drying in a vacuum oven at 4O ⁰ C overnight were 5:54 g of the desired compound. the mass findings of 304 confirmed the expected molecular weight. 1.6) Cu [H-Gly-Dab-Lys-OH] x HOAc x (H ₂ O)

2.5 g of the above peptide 1.5 were dissolved in 40 ml of WFI. The pH was 6.06. Thereafter, 0.658 g of Cu (OH) ₂ was added as a solid. The pH dropped to 5.34. For one hour, the pH was adjusted to 6.95 with 115 ml of NH ₄ OH solution (0.05 M). The resulting dark blue, slightly turbid solution was filtered and lyophilized. After additional drying overnight at 30 ⁰ C under vacuum g 2.67 of the desired compound were obtained, its molecular weight was confirmed by a mass finding of 365th

In an analogous manner, the compounds of Tables 1 and 2 can be prepared.

2. Preparation of a cosmetic cream

Disperse the Keltrol RD well in phase B.

Heat phases A and B separately to 80 ^° C.

While stirring, add phase A to phase B and homogenize.

Allow to cool to room temperature, add phase C, then adjust the pH to about 5.0 - 5.5 using phase D. 3. Biological activity

3.1 Determination of the stimulation of laminin V synthesis in keratinocyte cell cultures of the cell line HaCaT by treatment with the compounds according to the invention

The laminin V production per cell of in vitro cultured HaCaT keratinocytes was detected by an ELISA (Enzyme-Linked Immunosorbent Assay). In the presence of the compounds according to the invention, the increase in the laminin V production of the cells was quantified by this method.

The human HaCaT keratinocytes were a gift of Prof. Fusenig from the German Cancer Research Center in Heidelberg and were in

Culture medium grown according to standard cell culture methods. After 72

Hours of incubation with the respective active ingredients, the quantitative determination is carried out with a specific for laminin V.

Antibody. After determination of laminin V content, the cell number by means of the CyQÜANT ^® is the firm Molecular Probes determined. From the

Individual values are used to calculate the laminin V content per cell as units.

Material:

Culture medium: Test medium:

- DMEM - DMEM

- 10% FCS - no FCS

- 100 IU / ml penicillin - 100 IU / ml penicillin

- 0.1mg / ml streptomycin - 0.1mg / ml streptomycin wash buffer: milk solution:

- 0.05M Tris, pH 8.5 - wash buffer

- 0.15M NaCl - 5% milk powder

- 0.1% BSA

- 0.1% Tween-20

AK dilution solution: substrate solution:

- 50ml SuperBlock (37515; Pierce) - 1 ImmunoPure® OPD Tablet

(34006, Pierce)

- 450ml H ₂ O - 9ml H ₂ O - 0.05% Tween - ImI Stable Peroxide

Substr. Buffer, 10x (34062; Pierce)

1. AK (P3H9-2, Santa Cruz Biotechnology, Inc.) is diluted 1/6000 and 2. AK (31430; Socochim S.A.) 1/500 with AK-Verd.Lsg.

Method:

The keratinocytes are incubated at a density of approximately 5000 cells / well seeded in 96well-plates for 3 days until confluency in the culture medium (37 ^C C 10% CO ₂ ). The medium is triplicate to test medium with three different concentrations replaced. The following controls are tested on each plate:

Negative controls: positive controls:

A) A)

- with cells - with cells

- without l.AK; with 2.AK - with 1st and 2nd AK

B) B)

- without cells - with cells

- with 1st and 2nd AK - with 1st and 2nd AK

with lOng / ml TGF-β2

C) For each compound, a well without cells is tested to exclude the non-specific binding of the two AK.

The plates are incubated for a further 72 hours. Thereafter, the deposited laminin V is detected and quantified according to the following protocol:

■ Discard medium and wash with 200μl / well PBS

■ Fix with lOOμl / well methanol -> 15min / RT / Shaker 600rpm

■ Discard methanol and block with 200μl / well milk solution -> 30min / RT / Shaker 600rpm

■ discard milk solution and add lOOμl / well l.AK-Verd. incubate -> 2h / RT / Shaker 600rpm

■ l.AK-Verd. discard and wash 3x with 200μl / well wash buffer ^■ with 100μl / well 2.AK-Verd. incubate -> 3h / RT / Shaker 600rpm

^■ 2.AC-Verd. discard; Wash 3x with 200μl / well wash buffer and 1x 100μl / well PBS

■ add 100μl / well substrate solution -> 15min / RT / Shaker 600rpm

^■ Stop with 50μl / well H ₂ SO ₄ (2M) reaction and measure at 492nm.

^■ The staining solution is discarded, the plate is distilled twice with H ₂ O. washed and frozen for about 16 hours at -80 ⁰ C.

^■ The plate is thawed and the cell count is measured using the CyQUANT Assay according to the manufacturer's instructions.

The laminin V production per cell is calculated according to the following formula: (value OD _{LamInn v} / value RFU _Ze ii _z ahi) x 100

The calculated values are arbitrary units. The results for the compounds 1.1 to 1.11, 1.90, 2.1 to 2.11, and 2.90 of Tables 1 and 2, a better or comparable effect based on the reference substance H-Gly-His-Lys-OH.

3.2 Determination of the Stimulation of Collagen Synthesis Type I in Fibroblast Cell Cultures by Treatment with the Compounds According to the Invention.

Type I collagen of in vitro cultured skin fibroblasts was detected by an enzyme-linked immunosorbent assay (ELISA). In the presence of the active ingredients, the increase in collagen production of the cells was quantified by this method.

The human dermal fibroblasts were isolated from foreskin and grown in the culture medium.

After 72 h incubation period with the corresponding active ingredients, the quantitative determination is carried out with an antibody specific for collagen I.

Material:

Culture medium: Test medium:

- MEM - MEM - 10% FCS no FCS

- 100IU / ml penicillin 100IU / ml penicillin

- 0.1 mg / ml streptomycin 0. 1 mg / ml streptomycin

- IMM NEAA IMM NEAA

- ImM Na pyruvate ImM Na pyruvate

- 2mM L-glutamine 2mM L-glutamine

- 2OmM Hepes Buffer 2OmM Hepes Buffer

Wash Buffer: Milk solution:

- 0.05M Tris, pH 8.5 wash buffer

- 0.15M NaCl 5% milk powder

- 0.1% BSA

- 0.1% Tween-20

AK dilution solution: substrate solution:

- 50ml SuperBlock; (37515; Pierce) - 1 ImmunoPure® OPD Tablet

(34006, Pierce)

- 450ml H ₂ O - 9ml H ₂ O

- 0.05% Tween - ImI Stable Peroxide Substrate.

Buffer, 10x (34062; Pierce)

1. AK (MAB1340, Chemicon) and 2. AK (31430, Socochim S.A.) are diluted with AK-Verd.Lsg 1/500.

Method:

The fibroblasts are for 3 days at a density of approximately 5000 cells / well in 96-well plates to confluence in the culture medium were incubated (37 ⁰ C / 5% CO _2. The medium is replaced with assay medium with three different concentrations in triplicate to the test substance The following controls are tested on each plate:

Negative controls: positive controls:

A) A)

- with cells - with cells - without l .AK; with 2. AK - with 1. and 2. AK

B) B)

- without cells - with cells

- with 1st and 2nd AK - with 1st and 2nd AK

with lOng / ml TGF-β1

C) For each test substance, a well without cells is tested to exclude the non-specific binding of the two AK.

The plates are incubated for a further 72 hours. Thereafter, the deposited collagen I is detected and quantified according to the following protocol:

■ Discard medium and wash with 200μl / well PBS

■ fix with lOOμl / well methanol -> 15min / RT / shaker βOOrpm

■ discard milk solution and add lOOμl / well l.AK-Verd. incubate -> 2h / RT / Shaker βOOrpm

■ l.AK-Verd. discard and wash 3x with 200μl / well Washbuffer

■ with lOOμl / well 2.AK-Verd. incubate -> 3h / RT / Shaker βOOrpm

■ 2.AC-Verd. discard; Wash 3x with 200μl / well wash buffer and 1x100μl / well PBS

■ add 100μl / well substrate solution -> 20min / RT / Shaker 600rpm

^■ Stop with 50μl / well H ₂ SO ₄ (2M) reaction and measure at 492nm. The compounds 1.1 to 1.12, 1.91, 2.1 to 2.12, and 2.91 of Tables 1 and 2 show a better effect than for the reference substance H-Gly-His-Lys-OH.

3.3 Determination of stimulation of glycosaminoglycan (GAG) synthesis in fibroblasts

Normal human fibroblasts are cultured in 96well plates and mixed with the novel active substances in different Concentration incubated. After the incubation phase, the cell culture supernatant is assayed for the content of hyaluronic acid by means of an ELISA. The results for the compounds 1.1 to 1.11, 1.90, 2.1 to 2.11, and 2.90 of Tables 1 and 2, a better effect than for the reference substance H-Gly-His-Lys-OH.

Claims

claims

1. Biologically active tripeptides of the general formula (I)

wherein

R ^{1 is} hydrogen or C ¹ -C 6 -alkylcarbonyl,

R ^{2 is} 4-imidazolyl or -CH ₂ NH ₂ ,

R ^{3 is} hydrogen, Ci-Ci ₆ -alkyl or optionally substituted

Arylalkyl (C ₁ -C ₄ ), A -CH ₂ -, -CH ₂ CH ₂ -, C (CH ₃ ) ₂ , -NH-, or -CH (R ⁴ ) -, wherein the group [-CH (R ⁴ ) -] is D-configured; R ^{4 is} C 1 -C ₄ -alkyl or benzyl and X is oxygen (-O-) or -NH-;

Mean oxygen; still at the same time

A is -CH ₂ -, X is -NH- and R ^{3 is} hydrogen.

2. Compounds according to claim 1, wherein A is -CH ₂ -, -CH ₂ CH ₂ -, -NH- or -CH (R ⁴ ) -, wherein the group is [-CH (R ⁴ ) -] D- configured ,

3. Compounds according to claim 1 or 2, wherein not simultaneously A is -CH ₂ -, or -CH (R ⁴ ) -, wherein R ^{4 is} -CH ₂ -CH ₃ or -CH (CH ₃ ) ₂ , R ² 4- Imidazolyl and X oxygen mean.

4. Compounds according to any one of claims 1-3, wherein R ^{2 is} -CH ₂ NH ₂ .

5. Compounds according to any one of claims 1-4, wherein R ^{3 is} hydrogen and X is oxygen.

6. Compounds according to any one of claims 1-4, wherein R ^{3 is} benzyl and X is -NH-. ,

7. Use of the compounds of formula (I) according to any one of claims 1-6, their copper complexes; Salts of the tripeptides of the formula

(I) and salts of the 1: 1 complexes of these tripeptides with Cu ⁺² [Cu (II)], as dermatologically cosmetic agents, in particular for stimulating the synthesis of the extracellular matrix, preferably collagen I and III, the proteins of the basement membrane, preferably laminin V, for the accumulation of glycosaminoglycans, decorin and elastin.

8. Use of the compounds of formula (I) according to any one of claims 1-6, their copper complexes; Salts of the tripeptides of the formula

(I) and salts of 1: 1 complexes of these tripeptides with Cu ⁺² [Cu (II)], for the preparation of cosmetically dermatologically active compositions, in particular for the preparation of skin care agents, preferably for preventing the formation and reduction of wrinkles and against all Consequences of skin aging of natural endogenous or accelerated exogenous nature, in particular skin thickness, skin elasticity, skin contrast and skin hydration.

Cosmetically dermatologically active composition, characterized in that it contains at least one compound of the formula (I) according to one of Claims 1-6, the copper complexes thereof; at least one salt of the tripeptides of formula (I) and / or at least one salt of the 1: 1 complexes of these tripeptides with Cu ⁺² [Cu (II)] containing, preferably ppm in an amount ranging from 0.01 to I ¹ OOO ppm (w / w), in particular between 1 ppm and 100 ppm (w / w), calculated on the weight of the compound according to the invention and of the carrier material or carrier materials.

10. Cosmetically dermatologically active composition, characterized in that it a) at least one compound of formula (I) according to any one of claims 1-6, the copper complexes thereof and salts thereof, and b) a safe and effective amount of at least one additional skin care active selected from the group comprising: antisagging agents, anti -Aknewirkstoffe, vitamin B3 compounds, retinoids, di-, tri-, tetra-, penta- and hexapeptides and derivatives thereof, hydroxy acids, radical scavengers, anti-inflammatory agents, skin tanning agents, skin whitening agents, anti-cellulite agents, flavonoids, antimicrobial agents, skin remedies, antifungal drugs

Sunscreens, farnesol, phytantriol, allantoin, glucosamine and mixtures thereof.

11. A composition according to claim 10, characterized in that it contains a dermatological carrier.

12. A composition according to any one of claims 9-11, characterized in that it in the form of a solution, a dispersion, an emulsion or encapsulated in carriers, preferably in macro-, micro- or nanocapsules, in liposomes or chylomicrons, or enclosed in macro -, micro or nanoparticles or in

Microsponges or absorbed on powdered organic polymers, talc, bentonite and other mineral carriers.

13. A composition according to any one of claims 9-11, characterized in that these in the form of an emulsion, milk, lotion, ointment, a gelling and viscous, stress-active and emulsifying polymers, a Pommade, a shampoo, a soap, a gel, powder , Sticks or sticks, sprays, body oil, a face mask or patch for transdermal application.

14. A composition according to any one of claims 9-13 as a cosmetically active agent, in particular for the stimulation of the production of collagen I, laminin V, glycosaminoglycan, decorin and elastin in human skin, as a skin care agent, in particular for preventing the formation and reduction of Wrinkles and against all Consequences of skin aging of natural endogenous or accelerated exogenous species (helioderma, pollution).

15. A method for increasing the production of collagen V and / or laminin V and / or glycosaminoglycan and / or decorin and / or elastin in human skin and / or for the delay or treatment of skin aging, characterized in that a compound according to one of Claims 1-6 or a composition according to claims 9-14 applied to the skin.