U.S. patent application number 10/502627 was filed with the patent office on 2005-06-02 for use of a compound inactivating the kinase a protein in a composition containg a cosmetically acceptable medium in order to lighten the skin.
Invention is credited to Garcia, Christine, STOLTZ, Corinne.
Application Number | 20050118119 10/502627 |
Document ID | / |
Family ID | 27589581 |
Filed Date | 2005-06-02 |
United States Patent
Application |
20050118119 |
Kind Code |
A1 |
STOLTZ, Corinne ; et
al. |
June 2, 2005 |
Use of a compound inactivating the kinase a protein in a
composition containg a cosmetically acceptable medium in order to
lighten the skin
Abstract
A method of using a compound to lighten the skin by inactivating
protein kinase A. The compound may be in a composition with either
a pharmacologically or cosmetically acceptable medium. Depending
upon the medium and concentration of the compound, the composition
may be used either therapeutically or non-therapeutically.
Inventors: |
STOLTZ, Corinne; (Thiais,
FR) ; Garcia, Christine; (Castres, FR) |
Correspondence
Address: |
Air Liquide
Intellectual Property Department
2700 Post Oak Blvd., Suite 1800
Houston
TX
77056
US
|
Family ID: |
27589581 |
Appl. No.: |
10/502627 |
Filed: |
July 20, 2004 |
PCT Filed: |
January 22, 2003 |
PCT NO: |
PCT/FR03/00210 |
Current U.S.
Class: |
424/62 ;
514/18.6 |
Current CPC
Class: |
A61K 2800/782 20130101;
C07C 233/51 20130101; A61K 8/44 20130101; A61P 17/00 20180101; A61K
31/41 20130101; A61K 31/00 20130101; C07C 233/49 20130101; A61K
31/55 20130101; A61Q 17/04 20130101; A61Q 19/02 20130101 |
Class at
Publication: |
424/062 ;
514/009 |
International
Class: |
A61K 038/12; A61K
007/135 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 25, 2002 |
FR |
02/00925 |
Claims
1-26. (canceled)
27. A method of lightening skin comprising inactivating the protein
kinase A with a composition comprising: a) a cosmetically
acceptable medium; and b) a compound.
28. A method of lightening skin comprising inactivating the protein
kinase A with a composition comprising: a) a cosmetically
acceptable medium; and b) a compound of general formula (I): 7or
salts thereof, wherein: 1) R.sub.1 represents the characterizing
chain of a fatty acid comprising: i) about 3 to about 30 carbon
atoms; and ii) a characteristic comprising at least one member
selected from the group consisting of: aa) saturated; bb)
unsaturated; cc) linear; and dd) branched; 2) R.sub.2 represents
the characterizing chain of an amino acid; and 3) m is between
about 1 and about 50.
29. The method of claim 28, wherein said compound comprises a
radical of general formula (II): R.sub.1--C(.dbd.O)-- (II) wherein
said radical comprises about 7 to about 22 carbon atoms.
30. The method of claim 29, wherein said radical comprises at least
one member selected from the group consisting of: a) octanoyl; b)
decanoyl; c) undecylenoyl; d) dodecanoyl; e) tetradecanoyl; f)
hexadecanoyl; g) octadecanoyl; h) eicosanoyl; i) docosanoyl; j)
8-octadecenoyl; k) eicosenoyl; l) 13-docosenoyl; m)
9,12-octadecadienoyl; and n) 9,12,15-octadecatrienoyl radical.
31. The method of claim 30, wherein said radical comprises at least
one member selected from the group consisting of: a) octanoyl; b)
.omega.-undecylenoyl; c) dodecanoyl; d) hexadecanoyl; e)
8-octadecenoyl; f) 13-docosenoyl; g) 9,12-octadecadienoyl; and h)
9,12,15-octadecatrienoy- l.
32. The method of claim 31, wherein said R.sub.2 further comprises
at least one member selected from the group consisting of: a)
glycine; b) alanine; c) serine; d) aspartic acid; e) glutamic acid;
f) valine; g) threonine; h) arginine; i) lysine; j) proline
leucine; k) phenylalanine; l) isoleucine; m) histidine; n)
tyrosine; o) tryptophan; p) asparagine; q) glutamine; r) cysteine;
s) cystine; t) methionine; u) hydroxyproline; v) hydroxylysine; w)
sarcosine; and x) ornithine.
33. The method of claim 32, further comprising a residue, said
residue comprising at least one member selected from the group
consisting of: a) a residue of general formula (IIIa)
--HN--CH(R.sub.2)--C(.dbd.O)-- (IIIa), and b) a residue of general
formula (IIIb) 8
34. The method of claim 33, wherein said R.sub.2 comprises at least
one member selected from the group consisting of: a) phenylalanine;
b) tyrosine; c) histidine; d) methionine; e) cysteine; and f)
tryptophan.
35. The method of claim 34, wherein said m is less than about
5.
36. The method of claim 35, wherein said m is less than or equal to
about 2.
37. The method of claim 36, wherein said m is less than or equal to
about 1.4.
38. The method of claim 37, wherein said m is equal to about 1.
39. A method which may be used for treating skin comprising
lightening said skin non-therapeutically with a composition,
wherein said composition comprises: a) a cosmetically acceptable
medium; and b) an effective amount of at least one compound that
inactivates protein kinase A.
40. The method of claim 39, wherein said composition comprises
between about 0.01 % and about 10% of its total weight of said
compound.
41. The method of claim 40, wherein said composition comprises
between about 0.1% and about 5% of its total weight of said
compound.
42. The method of claim 41, wherein said composition comprises
between about 1% and about 5% of its total weight of said
compound.
43. A composition which may be used for lightening the skin
comprising: a) a pharmaceutically acceptable medium; and b) an
effective amount of at least one compound that inactivates protein
kinase A.
44. The composition of claim 43, wherein: a) said composition is
pharmaceutical; and b) said composition is suitable for therapeutic
use.
45. The method of claim 44, wherein said composition comprises
between about 0.01 % and about 10% of its total weight of said
compound.
46. The method of claim 45, wherein said composition comprises
between about 0.1% and about 5% of its total weight of said
compound.
47. The method of claim 46, wherein said composition comprises
between about 1% and about 5% of its total weight of said
compound.
48. The method of claim 27, wherein said compound also inactivates
adenylate cyclase.
49. The method of claim 33, wherein: a) said radical comprises at
least one member selected from the group consisting of: 1) octanoyl
radicals; and 2) .omega.-undecylenoyl radicals; and b) said residue
comprises at least one member selected from the group consisting
of: 9wherein said R.sub.2 represents the characterizing chain of
phenylalanine.
50. The method of claim 48, wherein: a) said radical comprises at
least one member selected from the group consisting of: 1) octanoyl
radicals; and 2) .omega.-undecylenoyl radicals; and b) said residue
comprises at least one member selected from the group consisting
of: 10wherein said R.sub.2 represents the characterizing chain of
phenylalanine.
51. The method of claim 42, wherein said compound also inactivates
adenylate cyclase.
52. The composition of claim 47, wherein said compound also
inactivates adenylate cyclase.
53. The method of claim 38, wherein said compound further comprises
an affinity for the melanocyte specific hormone (.alpha.-MSH)
receptor.
54. The method of claim 51, wherein said compound further comprises
an affinity for the melanocyte specific hormone (.alpha.-MSH)
receptor.
55. The composition of claim 52, wherein said compound further
comprises an affinity for the melanocyte specific hormone
(.alpha.-MSH) receptor.
56. A composition comprising N-(.omega.-undecylenoyl)phenylalanine
of formula (Ia): 11
57. The composition of claim 56, wherein said composition is used
cosmetics.
58. A pharmaceutical composition comprising: a) a pharmaceutically
acceptable medium; and b) an effective amount of the compound
N-(.omega.-undecylenoyl)phenylalanine of formula (Ia): 12
59. A composition comprising an emulsion, wherein said emulsion
comprises between about 0.01% and about 10% of its total weight,
the compound N-(.omega.-undecylenoyl)phenylalanine of formula (Ia):
13
60. The composition of claim 59, wherein said emulsion comprises
between about 0.1% and about 5% of its total weight of said
compound.
61. The composition of claim 60, wherein said emulsion further
comprises between about 1% and about 5% of its total weight of said
compound.
Description
[0001] The present invention relates to a novel use of cosmetic
active agents for lightening the skin.
[0002] Most of the commercially available depigmenting cosmetic
formulations are based on kojic acid, arbutin or magnesium ascorbyl
phosphate.
[0003] The inventors became interested in the development of novel
depigmenting active agents that have better compatibility with the
skin than those of the prior art. They demonstrated that molecules
that inactivate protein kinase A give rise to a skin depigmentation
that was attributed hitherto only to inhibition of the enzyme
phosphorylated tyrosinase.
[0004] Accordingly, according to a first aspect, the subject of the
invention is the use of a compound that inactivates protein kinase
A in a composition containing a cosmetically acceptable medium, for
lightening the skin.
[0005] The relationship between the skin-lightening activity and
the inactivation of protein kinase A (PKA) may be explained by the
following biochemical mechanism:
[0006] The inhibition of protein kinase A induces reduced
activation of tyrosinase, as a result of the reduced conversion of
the latter enzyme into phosphorylated tyrosinase; this reduced
activation of tyrosinase results in a reduction in melanin
synthesis, giving rise to skin depigmentation.
[0007] The expression "compound that inactivates protein kinase A"
especially denotes any compound which, by incubating protein kinase
A in the presence of adenosine triphosphate and a protein that may
be phosphorylated, for instance the histone H1, inhibits its
phosphorylation, with a percentage of inhibition of greater than or
equal to 10%, more particularly with a percentage of inhibition of
greater than or equal to 25% and preferably greater than or equal
to 50%.
[0008] A subject of the invention is, more particularly, the use of
a compound of formula (I): 1
[0009] or salts thereof, in which R.sub.1 represents the
characterizing chain of a saturated or unsaturated, linear or
branched fatty acid containing from 3 to 30 carbon atoms, R.sub.2
represents the characterizing chain of an amino acid and m is
between 1 and 50, or a mixture of said compounds of formula (I) or
salts thereof, in a composition containing a cosmetically
acceptable medium, for lightening the skin.
[0010] The compound of formula (I) as defined above may be in free
acid form or in partially or totally salified form. When the
compound of formula (I) is in salified form, the salts are
especially alkali metal salts such as the sodium, potassium or
lithium salts, alkaline-earth metal salts such as the calcium,
magnesium or strontium salts; the ammonium salt or the salt of an
amino alcohol, for instance the (2-hydroxyethyl)-ammonium salt.
They may also be metal salts such as divalent zinc or manganese
salts or trivalent iron, lanthanum, cerium or aluminum salts.
[0011] In the description hereinbelow, the expression compound of
formula (I) means the compound of formula (I) in free form or in
partially or totally salified form.
[0012] The expression "characterizing chain" used to define the
radicals R.sub.1 and R.sub.2 denotes the nonfunctional main chain
of the fatty acid or of the amino acid under consideration.
[0013] Thus, for a fatty acid corresponding to the general formula
R.sub.1--C(.dbd.O)--OH, the characterizing chain will be the chain
represented by R.sub.1.
[0014] The subject of the invention is, mainly, the use of a
compound of formula (I) as defined above, in which the group
R.sub.1--C(.dbd.O)-- contains from 7 to 22 carbon atoms.
[0015] R.sub.1--C(.dbd.O)-- especially represents an octanoyl,
decanoyl, undecylenoyl dodecanoyl, tetradecanoyl, hexadecanoyl,
octadecanoyl, eicosanoyl, docosanoyl, 8-octadecenoyl, eicosenoyl,
13-docosenoyl, 9,12-octadecadienoyl or 9,12,15-octadecatrienoyl
radical.
[0016] A subject of the invention is, more particularly, the use of
a compound of formula (I) as defined above, in which the fragment
R.sub.1--C(.dbd.O) is chosen from octanoyl, .omega.-undecylenoyl,
dodecanoyl, hexadecanoyl, 8-octa-decenoyl, 13-docosenoyl,
9,12-octadecadienoyl and 9,12,15-octadecatrienoyl radicals.
[0017] For an amino acid represented by the general formula
(IIIa):
H.sub.2N--CH(R.sub.2)--C(.dbd.O)--OH (IIIa)
[0018] and for a cyclic amino acid represented by formula (IIIb):
2
[0019] the characterizing chain will be the chain represented by
R.sub.2.
[0020] R.sub.2 especially represents the characterizing chain of an
amino acid chosen from glycine, alanine, serine, aspartic acid,
glutamic acid, valine, threonine, arginine, lysine, proline
leucine, phenylalanine, isoleucine, histidine, tyrosine,
tryptophan, asparagine, glutamine, cysteine, cystine, methionine,
hydroxyproline, hydroxylysine, sarcosine and ornithine.
[0021] The subject of the invention is, mainly, the use of a
compound of formula (I) as defined above, in which, in at least one
of the residues 3
[0022] R.sub.2 represents the characterizing chain of
phenyl-alanine, tyrosine, histidine, methionine, cysteine or
tryptophan.
[0023] A subject of the invention is, more particularly, the use of
a compound of formula (I) as defined above, in which m is a decimal
number between 1 and 10 and is preferably less than 5.
[0024] According to a most particular aspect of the present
invention, in formula (I) as defined above, m is less than or equal
to 2 and is more particularly less than or equal to 1.4.
[0025] According another most particular aspect of the present
invention, in formula (I) as defined above, m is equal to 1.
[0026] According to another particular variant of the present
invention, only one compound of formula (I), as defined above, is
used in the composition containing the cosmetically acceptable
medium.
[0027] According to another particular variant of the present
invention, a mixture of compounds of formula (I) as defined above
is used, and more particularly
[0028] either a mixture of compounds of formula (I) all comprising
the same fragment R.sub.1--C(.dbd.O),
[0029] or a mixture of compounds of formula (I) in which m is equal
to 1 and all comprising the same fragment 4
[0030] The compounds of formula (I) are generally obtained by
N-acylation of compounds of formula (IIIa) or (IIIb) as defined
above, or salts thereof.
[0031] When it is a mixture of compounds of formula (I), it is
obtained, for example, by N-acylation of the amino acid mixture
resulting from the total or partial hydrolysis of proteins of any
origin.
[0032] These proteins may be of animal origin, for instance
collagen, elastin, fish flesh protein, fish gelatin, keratin or
casein, of plant origin, for instance, proteins from cereals,
flowers or fruit, for instance proteins derived from soybean,
sunflower, oat, wheat, maize, barley, potato, lupin, bean, sweet
almond, kiwi, mango or apple; they may also be proteins obtained
from chorellae (unicellular algae), pink algae, yeasts or silk.
[0033] This hydrolysis is performed, for example, by heating a
protein placed in an acidic or alkaline medium to temperatures of
between 60 and 130.degree. C.
[0034] This hydrolysis may also be performed enzymatically with a
protease, optionally coupled to an alkaline or acidic
posthydrolysis. When m is greater than 1, R.sub.2 represents one
and the same chain or several chains characterizing different amino
acids, depending on the protein hydrolyzed and the degree of
hydrolysis.
[0035] The aminograms of a few proteins of plant origin are given
in the following table:
1 TABLE A Origin of the protein (amino acid proportions expressed
as weight %) Oat Soybean Wheat Sunflower Glycine 6.9 4.2 3.2 6.2
Alanine 5.9 4.2 2.6 4.8 Serine 5.6 5.1 1.7 5.1 Aspartic acid 16.2
11.7 3.4 10.6 Glutamic acid 28.3 19.1 37.9 23.6 Valine 2.9 5.0 4.2
4.8 Threonine 3.1 3.9 2.7 4.4 Arginine 6.6 7.8 3.7 8.4 Lysine 3.6
6.2 1.9 3.2 Proline 4.7 5.4 11.7 3.0 Leucine 6.4 8.1 7.1 6.4
Phenylalanine 1.4 5.0 5.4 4.3 Isoleucine 2.2 4.8 3.7 4.1 Histidine
1.7 2.6 2.4 2.0 Tyrosine 1.5 3.5 3.1 2.7 Methionine 1.2 1.2 1.6 1.8
Cysteine/cystine 1.9 1.5 1.9 1.9 Tryptophan -- 1.0 1.0 1.3 Origin
of the protein (amino acid proportions expressed as weight %) Lupin
Potato Bean Maize Glycine 0.9 4.8 4.0 2.4 Alanine 2.4 5.0 4.0 7.95
Serine 6.1 5.8 4.9 5.1 Aspartic acid 15.8 12.5 10.5 10.6 Glutamic
acid 8.0 11.5 16.8 23.6 Valine 7.9 7.1 4.5 4.8 Threonine 8.1 6.1
3.6 4.4 Arginine 16.1 5.0 9.21 8.4 Lysine 7.1 7.8 6.5 6.2 Proline
-- 5.1 4.4 3.0 Leucine 7.45 10.4 7.4 8.1 Phenylalanine 8.6 6.4 4.4
4.3 Isoleucine 8.7 6.1 3.9 4.1 Histidine -- 2.2 2.6 2.0 Tyrosine --
5.7 3.6 2.7 Methionine 0.6 2.4 0.8 1.8 Cysteine/cystine -- 1.6 1.7
1.9 Tryptophan 1.2 1.4 1.2 1.3 Ornithine 0.4 -- -- --
[0036] The acylation reaction is known to those skilled in the art.
It is described, for example, in the international patent
application published under the number WO 98/09611. It is performed
either on an amino acid or on an amino acid mixture. The acylating
agent generally consists of an activated derivative of a carboxylic
acid of formula R.sub.1C(.dbd.O)--OH, such as a symmetrical
anhydride of this acid or an acid halide, for instance the acid
chloride or acid bromide. It may also consist of a mixture of
activated derivatives of carboxylic acids derived from natural oils
or fats of animal or plant origin, such as coconut oil, palm kernel
oil, palm oil, soybean oil, rapeseed oil, maize oil, beef tallow,
spermaceti oil or herring oil.
[0037] A subject of the invention is also a nontherapeutic process
for treating the skin to lighten it, characterized in that a
composition containing a cosmetically acceptable medium and an
effective amount of at least one compound that inactivates protein
kinase A is applied thereto.
[0038] A subject of the invention is also a pharmaceutical
composition for performing a therapeutic skin treatment to lighten
it, characterized in that it contains a pharmaceutically acceptable
medium and an effective amount of at least one compound that
inactivates protein kinase A.
[0039] In the compositions defined above, the compound that
inactivates protein kinase A is generally used in an amount of
between 0.01% and 10% of their weight, more particularly between
0.1% and 5% of their weight and most particularly between 1% and 5%
of their weight.
[0040] According to another particular aspect, a subject of the
invention is the use as defined above, characterized in that the
compound that inactivates protein kinase A also inactivates
adenylate cyclase.
[0041] The relationship between the skin-lightening activity and
the inactivation of adenylate cyclase may be explained by the
following biochemical mechanism:
[0042] The inactivation of adenylate cyclase results in reduced
conversion of intracellular ATP into cyclic AMP; the reduction in
the level of cyclic AMP results in inhibition of protein kinase A
(PKA); the inhibition of protein kinase A induces reduced
activation of tyrosinase as a result of the reduced conversion of
said enzyme into phosphorylated tyrosinase; this reduced activation
of tyrosinase results in a reduction in melanin synthesis, giving
rise to the skin depigmentation.
[0043] The expression "compound that inactivates adenylate cyclase"
especially denotes, in the context of the present invention, any
compound which, by incubation of this enzyme in the presence of
adenosine triphosphate, inhibits its conversion into cyclic
adenosine mono-phosphate, with a percentage of inhibition of
greater than or equal to 10%, more particularly with a percentage
of inhibition of greater than or equal to 25% and preferably
greater than or equal to 50%.
[0044] The compounds that inactivate adenylate cyclase contained in
said composition are more particularly chosen from the compounds of
formula (I) as defined above or salts thereof, and most
particularly from the compounds of formula (I) as defined above in
which R.sub.1--C(.dbd.O) is chosen from octanoyl and
.omega.-undecylenoyl radicals and in which, in at least one of the
residues 5
[0045] R.sub.2 represents the characterizing chain of
phenylalanine.
[0046] A subject of the invention is also a process as defined
above, characterized in that a composition containing a
cosmetically acceptable medium and an effective amount of at least
one compound that inactivates protein kinase A and adenylate
cyclase, and also a pharmaceutical composition as defined above,
characterized in that it contains an effective amount of at least
one compound that inactivates protein kinase A and adenylate
cyclase, are applied to the skin.
[0047] According to another particular aspect, a subject of the
invention is the use as defined above, characterized in that the
compound that inactivates protein kinase A and adenylate cyclase is
a compound with affinity for the melanocyte specific hormone
(.alpha.-MSH) receptor.
[0048] The relationship between the skin-lightening activity and
the affinity for the .alpha.-MSH receptor may be explained by the
following biochemical mechanism:
[0049] The competition between the hormone .alpha.-MSH and the
molecule with affinity for the .alpha.-MSH receptor results in a
reduced level of binding of said hormone to the cell receptors; the
consequence of this competition is to inhibit the activity of
adenylate cyclase, which results in reduced conversion of
intracellular ATP into cyclic AMP; the reduction in the level of
cyclic AMP results in inhibition of the enzyme protein kinase A
(PKA); the inhibition of protein kinase A induces reduced
activation of tyrosinase as a result of the reduced conversion of
said enzyme into phosphorylated tyrosinase; this reduced activation
of tyrosinase results in a decrease in melanin synthesis, giving
rise to skin depigmentation. It is this set of successive
inhibitions that bears witness to the .alpha.-MSH-antagonist nature
of the compounds of the invention.
[0050] The expression "compound with affinity for the melanocyte
specific hormone, .alpha.-MSH, receptor", in the context of the
present invention, denotes any compound which displaces the
specific binding of a radioactive ligand, for instance nucleoside
diphosphate-.alpha.-melanocyt- e specific hormone
([.sup.125I]NDP-.alpha.-MSH) to the .alpha.-melanocyte specific
hormone (.alpha.-MSH) type 1 receptor, known as the MC1R receptor,
with a percentage of inhibition of greater than or equal to 10%,
more particularly with a percentage of inhibition of greater than
or equal to 25% and preferably greater than or equal to 50%.
[0051] The melanocyte specific hormone antagonists contained in
said composition are more particularly chosen from the compounds of
formula (I) as defined above, or salts thereof.
[0052] A subject of the invention is also a process as defined
above, characterized in that a composition containing a
cosmetically acceptable medium and an effective amount of at least
one compound that inactivates protein kinase A and adenylate
cyclase, which is a melanocyte specific hormone antagonist, and
also a pharmaceutical composition as defined above, characterized
in that it contains an effective amount of at least one compound
that inactivates protein kinase A and adenylate cyclase, which is a
melanocyte specific hormone antagonist, are applied to the
skin.
[0053] As shown by the following examples, the compounds used in
the cosmetic or therapeutic treatments defined above are
characterized, unexpectedly, by skin-lightening activity that is
higher than that of the compositions of the prior art. They are
thus generally suitable for treatments for lightening the skin,
especially by depigmentation, and more particularly for removing or
attenuating colored marks appearing on elderly skin.
[0054] The compositions used in said treatments are generally in
the form of dilute aqueous or aqueous-alcoholic solutions, in the
form of simple or multiple emulsions, such as water-in-oil (W/O),
oil-in-water (O/W) or water-in-oil-in-water (W/O/W) emulsions, in
which the oil is of plant or mineral nature, or in the form of
powder. They may also be dispersed or impregnated onto fabric or
nonwoven materials, whether they are wipes, paper towels or
clothing.
[0055] The compositions used in said treatments are administered to
the individual in the conventional forms used in cosmetics and
pharmacy; these are more particularly topical, oral or parenteral
administrations.
[0056] In general, the compounds of formula (I) that inactivate
protein kinase A, possibly adenylate cyclase and possibly
melanocyte specific hormone antagonists, which are used in the
invention that is the subject of the present patent application, as
defined above, are combined with numerous types of adjuvants or
active principles used in cosmetic formulations, whether they are
fatty substances, organic solvents, thickeners, gelling agents,
softeners, antioxidants, opacifiers, stabilizers, foaming agents,
fragrances, ionic or nonionic emulsifiers, fillers, sequestering
agents, chelating agents, preserving agents, chemical screening
agents or mineral screening agents, essential oils, dyestuffs,
pigments, hydrophilic or lipophilic active agents, humectants, for
instance glycerol, preserving agents, dyes, fragrances, cosmetic
active agents, mineral or organic sunscreens, mineral fillers, for
instance iron oxides, titanium oxides and talc, synthetic fillers,
for instance Nylons and crosslinked or noncrosslinked poly(methyl
methacrylate), silicone elastomers, sericites or plant extracts, or
alternatively lipid vesicles, or any other ingredient usually used
in cosmetics.
[0057] As examples of oils that may be combined with the compound
of formula (I), mention may be made of paraffins, isoparaffins,
white mineral oils, plant oils, animal oils, synthetic oils,
silicone oils and fluoro oils; and more particularly:
[0058] oils of plant origin, such as sweet almond oil, coconut oil,
castor oil, jojoba oil, olive oil, rapeseed oil, groundnut oil,
sunflower oil, wheatgerm oil, maize germ oil, soybean oil,
cottonseed oil, alfalfa oil, poppy oil, pumpkin oil, evening
primrose oil, millet oil, barley oil, rye oil, safflower oil,
candlenut oil, passionflower oil, hazelnut oil, palm oil, shea
butter, apricot kernel oil, beauty-leaf oil, sysymbrium oil,
avocado oil or calendula oil;
[0059] ethoxylated plant oils;
[0060] oils of animal origin, such as squalene or squalane;
[0061] mineral oils, such as liquid paraffin, liquid petroleum
jelly and isoparaffins;
[0062] synthetic oils, especially fatty acid esters such as butyl
myristate, propyl myristate, cetyl myristate, isopropyl palmitate,
butyl stearate, hexadecyl stearate, isopropyl stearate, octyl
stearate, isocetyl stearate, dodecyl oleate, hexyl laurate,
propylene glycol dicaprylate, esters derived from lanolic acid,
such as isopropyl lanolate, isocetyl lanolate, fatty acid
monoglycerides, diglycerides and triglycerides, for instance
glyceryl triheptanoate, alkylbenzoates, poly-.alpha.-olefins,
polyolefins, for instance polyisobutene, synthetic isoalkanes, for
instance isohexadecane or isododecane, perfluoro oils and silicone
oils. Among the silicone oils, mention may be made more
particularly of dimethylpolysiloxanes, methylphenylpolysiloxanes,
silicones modified with amines, silicones modified with fatty
acids, silicones modified with alcohols, silicones modified with
alcohols and fatty acids, silicones modified with polyether groups,
modified epoxy silicones, silicones modified with fluoro groups,
cyclic silicones and silicones modified with alkyl groups.
[0063] As other fatty materials that may be combined with this
active agent, mention may be made of fatty alcohols or fatty
acids.
[0064] Among the thickening and/or emulsifying polymers used in the
present invention, there are, for example, homopolymers or
copolymers of acrylic acid or of acrylic acid derivatives,
acrylamide homopolymers or copolymers, homopolymers or copolymers
derived from acrylamide, homopolymers or copolymers of
acrylamidomethylpropanesulfonic acid, of vinyl monomer or of
trimethylaminoethyl acrylate chloride, sold under the names
Carbopol.TM., Ultrez.TM. 10, Permulen.TM. TR1, Permulen.TM. TR2,
Simulgel.TM. A, Simulgel.TM. NS, Simulgel.TM. EPG, Simulgel.TM. EG,
Luvigel.andgate. EM, Salcare.TM. SC91, Salcare.TM. SC92,
Salcare.TM. SC95, Salcare.TM. SC96, Flocare.TM. ET100,
Hispagel.TM., Sepigel.TM. 305, Sepigel.TM. 501, Sepigel.TM. 502,
Flocare.TM. ET58 and Stabileze.TM. 06; hydrocolloids of plant or
biosynthetic origin, for instance xanthan gum, karaya gum,
carrageenates or alginates; silicates; cellulose and its
derivatives; starch and its hydrophilic derivatives;
polyurethanes.
[0065] Among the waxes that may be used in the context of the
present invention, examples that may be mentioned include beeswax;
carnauba wax; candelilla wax; ouricury wax; Japan wax; cork fiber
wax or sugarcane wax; paraffin waxes; lignite waxes;
microcrystalline waxes; lanolin wax; ozokerite; polyethylene wax;
hydrogenated oils; silicone oils; plant waxes; fatty alcohols and
fatty acids that are solid at room temperature; glycerides that are
solid at room temperature.
[0066] Among the emulsifiers that may be used in the context of the
present invention, examples that may be mentioned include fatty
acids; ethoxylated fatty acids; fatty acid esters of sorbitol;
ethoxylated fatty acid esters; polysorbates; polyglycerol esters;
ethoxylated fatty alcohols; sucrose esters; alkylpolyglycosides;
sulfated and phosphated fatty alcohols or mixtures of
alkylpolyglycosides and of fatty alcohols described in French
patent applications 2 668 080, 2 734 496, 2 756 195, 2 762 317, 2
784 680, 2 784 904, 2 791 565, 2 790 977, 2 807 435 and 2 804
432.
[0067] As examples of active principles that may be combined with
the compound of formula (I) in order to synergistically potentiate
its properties, mention may be made of compounds with lightening or
depigmenting activity, for instance arbutin, kojic acid,
hydroquinone, ellagic acid, vitamin C, magnesium ascorbyl
phosphate, polyphenol extracts, grape extracts, pine extracts, wine
extracts, olive extracts, pond extracts, N-acyl proteins, N-acyl
peptides, N-acylamino acids, partial hydrolyzates of N-acyl
proteins, amino acids, peptides, total protein hydrolyzates,
partial protein hydrolyzates, polyols (for instance glycerol,
butylene glycol, etc.), urea, pyrrolidonecarboxylic acid or
derivatives of this acid, glycyrrhetinic acid, .alpha.-bisabolol,
sugars or sugar derivatives, polysaccharides or derivatives
thereof, hydroxy acids, for instance lactic acid, vitamins, vitamin
derivatives, for instance retinol, vitamin E and its derivatives,
minerals, enzymes, coenzymes, for instance coenzyme Q10, hormones
or "hormone-like" substances, soybean extracts, for instance
Raffermine.TM., wheat extracts, for instance Tensine.TM. or
Gliadine.TM., plant extracts, such as tannin-rich extracts,
isoflavone-rich extracts or terpene-rich extracts, extracts of
freshwater or seawater algae, essential waxes, bacterial extracts,
minerals, lipids in general, lipids such as ceramides or
phospholipids, active agents with slimming activity, for instance
caffeine or its derivatives, active agents with antimicrobial
activity or with purifying action on greasy skin, such as
Lipacide.TM. PVB, active agents with an energizing or stimulatory
property, for instance Sepitonic.TM. M3 or Physiognyl.TM.,
panthenol and its derivatives, for instance Sepicap.TM. MP,
antiaging active agents, for instance Sepilift.TM. DPHP,
Lipacide.TM. PVB, Sepivinol.TM. or Sepivital.TM., moisturizing
active agents, for instance Sepicalm.TM. S, Sepicalm.TM. VG and
Lipacide.TM. DPHP, "anti-photoaging" antiaging active agents,
active agents for protecting the integrity of the dermo-epidermal
junction, and active agents for increasing the synthesis of
components of the extracellular matrix.
[0068] As sunscreens that may be incorporated into the composition
according to the invention, mention may be made of any of those
featured in the Cosmetic Directive 76/768/EEC amended appendix
VII.
[0069] According to a final aspect of the present invention, a
subject thereof is N-(-.omega.-undecylenoyl)phenylalanine of
formula: 6
[0070] its cosmetic use, pharmaceutical compositions containing it
and emulsions characterized in that they have a content thereof of
between 0.01% and 10% of their weight, more particularly between
0.1% and 5% of their weight and most particularly between 1% and 5%
of their weight.
[0071] The experimental study that follows illustrates the
invention without, however, limiting it.
[0072] In vitro Evaluation of the Depigmenting Activity of
Undecylenoylphenylalanine
[0073] The object of this study was to demonstrate the depigmenting
activity of N-undecylenoylphenylalanine, according to a mechanism
involving the antagonist effect of the molecule on the a-melanocyte
specific hormone (.alpha.-MSH) type 1 receptor, known as the MC1R
receptor. This type of pharmacological receptor is mainly found in
the melanocytes.
[0074] The control of melanogenesis using this receptor is shown in
FIG. 1. It especially involves adenylate cyclase, cAMP, protein
kinase A and tyrosinase. By binding to the receptor MC1R,
.alpha.-MSH stimulates the .alpha. subunit of the stimulating
protein G (Prot G.alpha.S). This protein activates the enzyme
adenylate cyclase, which converts adenosine triphosphate (ATP) into
cyclic adenosine monophosphate (cAMP). The cAMP activates the A
protein kinases (PK A), which convert tyrosinase into
phosphorylated tyrosinase, which stimulates melanogenesis.
[0075] In a first step, the study thus consisted in evaluating the
binding capacities of N-undecylenoylphenylalanine to the receptors
MC1R, found in the melanocytes.
[0076] In a second step, the effect of N-undecylenoylphenylalanine
on the activities of adenylate cyclase, protein kinase A and
tyrosinase was evaluated.
[0077] In a third step, the depigmenting activity of
N-undecylenoylphenylalanine on melanocyte cultures of the B16/F1
line was determined in vitro by measuring the intracellular and
extracellular melanin contents and by measuring the tyrosinase
activity.
[0078] In a fourth step, the depigmenting activity of
N-undecylenoylphenylalanine was evaluated in a model of pigmented
reconstructed human epidermides (photo-type IV) in order to test
the efficacy of the product under real application conditions
(topical application of the formulated product).
[0079] The effects of the product were compared with those observed
in the case of various reference depigmenting products,
hydroquinone, kojic acid and arbutin.
[0080] 1--Affinity Study on MC1R Receptors
[0081] The affinity of N-undecylenoylphenylalanine, kojic acid and
arbutin was compared.
[0082] MC1R receptors are isolated from cell membranes of mouse
melanocytes of the B16/F1 line via the method described in:
Siegrist W., Oestreicher M., Stutz M., Girard J. and Eberle A. E.;
J. Recep. Res., 8, 1988, 323-343".
[0083] N-Undecylenoylphenylalanine, arbutin and kojic acid are
diluted to a concentration of 10 mg/ml in decinormal aqueous sodium
hydroxide solution. They are each tested separately at
concentrations of 0.1 mg/ml and 1 mg/ml. Sodium hydroxide has no
effect on the parameter studied.
[0084] The MC1R receptors are incubated, in the presence or absence
of these products, with an iodine-125 labeled radioactive ligand,
the nucleoside diphosphate-.alpha.-melanocyte specific hormone
[.sup.125I]NDP-.alpha.-MSH at a concentration of 0.05 nM, for 90
minutes at 22.degree. C.
[0085] Control cultures are incubated, in the absence of product,
and in the presence of the radioactive ligand. Each test is
performed in triplicate.
[0086] After incubation for 90 minutes, the cell membranes are
rapidly filtered and the filters are washed several times with cold
buffer. The amount of radioactive ligand bound to the MC1R
receptors is measured using a scintillation counter (Topcount,
Packard).
[0087] The results given in the table below are the means of the
three tests performed for each of the products. They are expressed
as a percentage of specific binding relative to the control group
and as a percentage of inhibition of this binding.
2 Inhibition of specific Activity relative to binding by the test
Test the control products products at 0.1 mg/ml at 1 mg/ml at 0.1
mg/ml at 1 mg/ml Arbutin 100.80 .+-. 0.55 96.30 .+-. 4.16 0% 3.7%
Kojic acid 104.50 .+-. 1.38 124.00 .+-. 1.87 0% 0% N-Undecyl- 57.70
.+-. 2.38 4.20 .+-. 0.86 42.3% 95.8% enoylphenyl- alanine
[0088] The results demonstrate that at the test concentrations,
neither arbutin nor kojic acid, which are the reference
depigmenting compounds, displaces the specific binding of the
ligand, .sup.125I]NDP-.alpha.-MSH; in contrast,
N-undecylenoylphenylalanine displaces 42% and 96%, respectively, of
the binding of [.sup.125I]NDP-.alpha.-MSH to the MC1R
receptors.
[0089] 2--Study of Adenylate Cyclase Activation
[0090] The influence of N-undecylenoylphenylalanine, kojic acid and
arbutin on the conversion of ATP into cAMP was compared via a
radioimmunological assay.
[0091] Adenylate cyclase, which converts ATP into cAMP, is
extracted from rat brains via the method described in "Salamon Y.,
Londos C. and Rodbell M.; Anal. Biochem., 58, 1974, 541-548"; it is
then activated with 10 .mu.M of forskolin.
[0092] N-Undecylenoylphenylalanine, arbutin and kojic acid are
diluted to a concentration of 10 mg/ml in decinormal aqueous sodium
hydroxide solution. They are each tested separately at a
concentration of 1 mg/ml. Sodium hydroxide has no effect on the
parameter studied.
[0093] The activated enzyme is incubated, in the presence or
absence of these products, and in the presence of 0.5 mM of ATP,
for 30 minutes at 30.degree. C.
[0094] Control cultures are incubated, in the absence of product,
and in the presence of ATP. Each test is performed in
triplicate.
[0095] After incubation for 30 minutes, the amount of cAMP produced
is evaluated via a radioimmunological assay performed using a
commercial kit; the radioactivity is measured with a scintillation
counter (Topcount, Packard), a small radioactivity count reflecting
small activation of adenylate cyclase.
[0096] The results given in the table below are the means of the
three tests performed for each of the products. They are expressed
as a percentage of enzymatic activity relative to the control group
and as a percentage of inhibition.
3 Enzymatic activity Inhibition of relative to the adenylate
cyclase Test products control activity Arbutin 109.7 .+-. 4.6% 0%
Kojic acid 45.0 .+-. 6.5% 55% N-Undecylenoylphenylalanine -16.0
.+-. 0.5% 100%
[0097] The results demonstrate that at 1 mg/ml, whereas arbutin has
no effect on this enzyme, kojic acid induces a moderate effect and
N-undecylenoylphenylalanine induces total inactivation.
[0098] 3--Study of Protein Kinase A Activity
[0099] The influence of N-undecylenoylphenylalanine, kojic acid and
arbutin on the phosphorylation of tyrosinase with protein kinase A
(PK A) was compared.
[0100] Protein kinase A is extracted from bovine brains via the
method described in: "Chijiwa T., Mishima A., Hagiwara M., Sano M.,
Hayashi K., Inoue T., Naito K., Shioka T., Hidaka H.; J. Biol.
Chem., 265, 1990, 5267-5272". It is then activated with 3 .mu.M of
cAMP.
[0101] N-Undecylenoylphenylalanine, arbutin and kojic acid are
diluted to a concentration of 10 mg/ml in decinormal aqueous sodium
hydroxide solution. They are each tested separately at a
concentration of 1 mg/ml. Sodium hydroxide has no effect on the
parameter studied.
[0102] The activated enzyme is incubated, in the presence or
absence of these products, and in the presence of .sup.33P-labeled
radioactive ATP ([.gamma.-.sup.33P]ATP) and 200 .mu.g/ml of histone
H.sub.1, for 20 minutes at 30.degree. C.
[0103] Control cultures are incubated, in the absence of product,
and in the presence of radioactive ATP and histone H.sub.1. Each
test is performed in triplicate.
[0104] After incubation for 20 minutes, the amount of
.sup.33P-labeled phosphorylated histone H.sub.1 is measured using a
scintillation counter (Topcount, Packard), a small radioactivity
count reflecting small activation of the protein kinase A.
[0105] The results given in the table below are the means of the
three tests performed for each of the products. They are expressed
as a percentage of enzymatic activity relative to the control group
and as a percentage of inhibition.
4 Enzymatic activity Inhibition of relative to the protein kinase A
Test products control activity Arbutin 90.9 .+-. 8.4% 9.1% Kojic
acid 113.3 .+-. 5.0% 0% N-Undecylenoylphenylalanine -0.4 .+-. 0.3%
100%
[0106] The results demonstrate that at 1 mg/ml,
N-undecylenoylphenylalanin- e totally inhibits the protein kinase A
activity, in contrast to kojic acid or arbutin.
[0107] 4--Study of Phosphorylated Tyrosinase Activity
[0108] The influence of N-undecylenoylphenylalanine, hydroquinone,
kojic acid and arbutin on the activity of phosphorylated tyrosinase
was compared by measuring the conversion of L-tyrosine into L-dopa
and dopaquinone, which is a colored product that can be quantified
via spectrophotometry (at 490 nm).
[0109] The tyrosinase used is a commercial product extracted from
fungi.
[0110] N-Undecylenoylphenylalanine, hydroquinone, arbutin and kojic
acid are diluted to a concentration of 10 mg/ml in decinormal
aqueous sodium hydroxide solution. They are each tested separately
at concentrations of 0.1 mg/ml and 1 mg/ml. Sodium hydroxide has no
effect on the parameter studied.
[0111] Tyrosinase at 66.66 IU/ml is incubated, in the presence or
absence of these products, and in the presence of 0.2 mM of
tyrosine, for 10 minutes at 37.degree. C.
[0112] Control cultures are incubated, in the absence of product,
and in the presence of tyrosinase and L-tyrosine. Each test is
performed in triplicate.
[0113] After incubation for 10 minutes, the amount of dopaquinone
histone formed is measured using a spectro-photometer at 490
nm.
[0114] The results given in the table below are the means of the
three tests performed for each of the products.
[0115] They are expressed as IU/1 of tyrosinase activity and as a
percentage of inhibition of the enzymatic activity relative to the
control.
5 Percentage of inhibition of tyrosinase activity with the test
products relative to the control Test products at 0.1 mg/ml at 1
mg/ml Hydroquinone 78 80 Arbutin 73 80 Kojic acid 76 80
N-Undecylenoylphenylalanine 80 100
[0116] The results demonstrate that at concentrations of 0.1 mg/ml
and 1 mg/ml, all the test products significantly inhibit the
activity of tyrosinase. However, the inhibitory activity of
undecylenoylphenylalanine is greater than that of the other test
products.
[0117] 5--Study of the Depigmenting Activity in B16/F1 Melanocyte
Cultures
[0118] The influence of N-undecylenoylphenylalanine, hydroquinone,
kojic acid and arbutin on the production of intracellular melanin
and extracellular melanin, in B16/F1 melanocyte cultures and on the
activity of phosphorylated tyrosinase, was compared.
[0119] Mouse melanocytes of the B16/F1 line are inoculated in
96-well culture plates at a density of 1500 cells/well. The cells
are cultured in a culture medium (MCM medium) at 37.degree. C.
under a humid atmosphere containing 5% CO.sub.2. The cells are used
at 60% of confluence, i.e. 4 days after inoculation.
[0120] The MCM medium has the following composition: DMEM medium
(Dulbecco's Modified Eagle's Medium) containing 4.5 g/l of glucose
supplemented with L-glutamine (2 mM), penicillin (50 IU/ml),
streptomycin (50 .mu.g/ml) and fetal calf serum (10% v/v).
[0121] N-Undecylenoylphenylalanine is diluted to 4 mg/ml in
decinormal aqueous sodium hydroxide solution. It is tested at 40
.mu.g/ml in the MCM medium. Sodium hydroxide has no effect on the
parameters analyzed.
[0122] Hydroquinone is tested at 5 .mu.g/ml in the MCM medium.
Given its toxicity, it is not tested at 40 .mu.g/ml.
[0123] Arbutin and kojic acid are tested at 40 .mu.g/ml in the MCM
medium.
[0124] The melanocyte cultures are incubated in the presence of the
test product or of the reference products for 72 hours at
37.degree. C., under a humid atmosphere containing 5% CO.sub.2.
[0125] Control cultures are incubated, in the absence of product,
in the MCM medium. These control cultures are prepared on each
culture plate.
[0126] Each test is performed six times.
[0127] 5.1--Measurement of the Extracellular Melanin Content
[0128] After incubation for 72 hours, the incubation media of the
cells (n=6) are taken up and stored at -80.degree. C. until the
time of evaluation of the effects. The extracellular melanin is
quantified by spectrophotometry at 450 nm. A melanin calibration
range is prepared in parallel.
[0129] The results are expressed as .mu.g/ml of extracellular
melamin and as a percentage of inhibition relative to the control
group.
6 Extracellular Inhibition of melanin extracellular (control:
melanin Test products 43 .+-. 11 .mu.g/ml) production Hydroquinone
at 5 .mu.g/ml 7 .+-. 1 .mu.g/ml 85% Arbutin at 40 .mu.g/ml 23 .+-.
6 .mu.g/ml 47% Kojic acid at 40 .mu.g/ml 13 .+-. 2 .mu.g/ml 70%
N-Undecylenoylphenylalanine 12 .+-. 1 .mu.g/ml 72% at 40
.mu.g/ml
[0130] 5.2--Measurement of the Intracellular Melanin Content
[0131] After incubation for 72 hours, a portion of the cell carpet
(n=3) is rinsed with phosphate-buffered saline (PBS; pH=7.4), the
composition of which is as follows: NaCl: 8 g/l, Na.sub.2HPO.sub.4:
1.15 g/l; KH.sub.2PO.sub.4: 0.2 g/l; KCl: 0.2 g/l; CaCl.sub.2: 0.1
g/l; MgCl.sub.2: 0.1 g/l. The intracellular melanin is solubilized
by incubation, with stirring, for 30 minutes at room temperature in
the presence of decanormal sodium hydroxide.
[0132] The intracellular melanin is quantified by spectrophotometry
at 450 nm. A melanin calibration range is prepared in parallel.
[0133] The results are expressed as .mu.g/ml of intracellular
melanin and as a percentage of inhibition relative to the control
group.
7 Intracellular melanin Inhibition of obtained intracellular
(control: melanin Test products 20 .+-. 4 .mu.g/ml) production
Hydroquinone at 5 .mu.g/ml 0.2 .+-. 0.1 .mu.g/ml 100% Arbutin at 40
.mu.g/ml 16 .+-. 2 .mu.g/ml 19% Kojic acid at 40 .mu.g/ml 17 .+-. 1
.mu.g/ml 17% N-Undecylenoylphenylalanine 7 .+-. 3 .mu.g/ml 66% at
40 .mu.g/ml
[0134] 5.3--Measurement of the Phosphorylated Tyrosinase
Activity
[0135] After incubation for 72 hours, the second portion of the
cell carpet (n=3) is rinsed with PBS. The cells are lyzed with
Triton.TM. X100 at a concentration of 0.1% (w/v) for 30 minutes at
room temperature. The activity of the endogenous tyrosinase is
evaluated by adding 0.1% (w/v) of L-dopa, followed by incubation
for 3 hours at 37.degree. C. in the absence of air and light. The
dopaquinone formed by the reaction between the tyrosinase and the
L-dopa is measured by spectrophotometry at 450 nm. A calibration
range of purified tyrosinase is prepared in parallel.
[0136] The results are expressed as IU/ml of tyrosinase activity
and as a percentage of inhibition relative to the control
group.
8 Tyrosinase activity Inhibition of (control: tyrosinase Test
products 9.8 .+-. 0.3 IU/ml) activity Hydroquinone at 5 .mu.g/ml
3.0 .+-. 0.3 IU/ml 69% Arbutin at 40 .mu.g/ml 7.6 .+-. 1.1 IU/ml
23% Kojic acid at 40 .mu.g/ml 6.8 .+-. 0.1 IU/ml 31%
N-Undecylenoylphenylalanine 3.2 .+-. 0.6 IU/ml 67% at 40
.mu.g/ml
[0137] 5.4--Measurement of the Intracellular Protein Content
[0138] This assay allows the cytotoxicity of the test products to
be evaluated. It is performed in cell lysates prepared as described
in the preceding paragraph.
[0139] The protein assay is performed according to the Coomassie
blue method described by: "Bradford M.; Anal. Biochem., 72, 1976,
248-254". The measurement is performed by spectrophotometry at 640
nm. A bovine serum albumin (BSA) calibration range is prepared in
parallel.
[0140] The results are expressed as mg/ml of proteins and as a
percentage of inhibition relative to the control group.
9 Total proteins Inhibition (control: of protein Test products 0.45
.+-. 0.01) quantity Hydroquinone at 5 .mu.g/ml 0.28 .+-. 0.01 mg/ml
38% Arbutin at 40 .mu.g/ml 0.43 .+-. 0.01 mg/ml 5% Kojic acid at 40
.mu.g/ml 0.41 .+-. 0.02 mg/ml 10% N-Undecylenoylphenylalanine 0.38
.+-. 0.01 mg/ml 17% at 40 .mu.g/ml
[0141] 5.5--Results Analysis
[0142] After incubation for 72 hours, hydroquinone, tested at 5
.mu.g/ml, inhibits the extracellular melanin content by 85%, the
intracellular melanin content by 100% and the tyrosinase activity
by 69%, respectively. However, the depigmenting effect of
hydroquinone is partly derived from its cytotoxic effect, since a
38% decrease in the total protein quantity is observed.
[0143] Arbutin, tested at 40 .mu.g/ml, inhibits the extracellular
melanin content by 47%, the intracellular melanin content by 19%
and the tyrosinase activity by 23%, respectively. At this
concentration, arbutin has no effect on the total protein
content.
[0144] Kojic acid, tested at 40 .mu.g/ml, inhibits the
extracellular melanin content by 70%, the intracellular melanin
content by 17% and the tyrosinase activity by 31%, respectively. At
this concentration, kojic acid has no significant effect on the
total protein content.
[0145] N-Undecylenoylphenylalanine, tested at 40 .mu.g/ml, inhibits
the extracellular melanin content by 72%, the intracellular melanin
content by 66% and the tyrosinase activity by 67%, respectively. At
this concentration, N-undecylenoylphenylalanine decreases the total
protein content by 17%.
[0146] N-Undecylenoylphenylalanine thus has depigmenting activity,
demonstrated by a concommitant reduction of the intracellular and
extracellular melanin contents and of tyrosinase activity. Unlike
that of hydroquinone, its depigmenting activity is not linked to a
cytotoxic effect. It has higher depigmenting activity than arbutin
and kojic acid.
[0147] 6--Study of the Depigmenting Activity in Reconstructed Human
Epidermides
[0148] The influence of N-undecylenoylphenylalanine, hydroquinone,
kojic acid and arbutin on the production of intracellular melanin
and extracellular melanin, in B16/F1 melanocyte cultures and on the
staining of epidermides, was compared.
[0149] Pigmented human epidermides (phototype IV) supplied by
Skinethic, of 0.63 cm.sup.2, are reconstructed from a coculture of
normal human keratinocytes (skin of the forearm, 3 year old donor,
2nd passage) and from normal human melanocytes (skin of the
forearm, 4 year old donor of phototype IV, 3rd passage). The
keratinocyte/melanocyte ratio is 10:1. The cocultures are
inoculated onto inert polycarbonate filters. They are cultured for
10 days in the medium supplied by Skinethic, consisting of MCDB 153
medium supplemented with 5 .mu.g/ml of insulin, 1.5 mM of calcium
and growth factors.
[0150] For these tests, the products are tested after having been
incorporated into a cosmetic formulation consisting of an emulsion
comprising an aqueous phase, 10% by weight of a fatty phase
(Lanol.TM. 1688), 2% by weight of an emulsifier (Simugel.TM. EG),
0.5% by weight of preserving agents (0.3% of Sepicide.TM. HB+0.2%
of Sepicide.TM. CI). After incorporation of the active principle,
the formulation is adjusted to pH=5.5.
[0151] N-Undecylenoylphenylalanine, arbutin and kojic acid are
incorporated therein at elevated temperature (75.degree. C.), in a
proportion of 1% or 3% by weight per unit volume (w/v).
[0152] On account of its toxicity, hydroquinone is incorporated
therein at a concentration of 0.1% by weight per unit volume
(w/v).
[0153] The epidermides are cultured in 6-well plates containing 1
ml of the medium described above. They are incubated at 37.degree.
C. under a humid atmosphere containing 5% CO.sub.2.
[0154] The formulations containing the various active principles
are applied to the surface of the epidermides, at a rate of 2
.mu.l/epidermis, using a sterile bacteriological inoculator. The
application is performed every day for 4 consecutive days. The
incubation medium of the reconstructed epidermides is renewed every
day for 4 consecutive days.
[0155] Control epidermides are treated with a formulation free of
active principle. Each test is performed in duplicate.
[0156] 6.1--Chromametric Measurement of the Epidermal
Pigmentation
[0157] Three days after the final topical application, the color of
the epidermides is evaluated using a chromameter (Minolta) by
measuring the following parameters L*, a* and b*:
[0158] L* is the lightness index. A depigmenting product should
increase this parameter;
[0159] a* represents the color spectrum from blue to green. A
depigmenting product should reduce this parameter;
[0160] b*: represents the color spectrum from red to yellow. A
depigmenting product should reduce this parameter.
[0161] The results are expressed in arbitrary units (AU) of each
parameter and as a percentage of the control group.
10 L* a* b* Control: Control: Control: 41.38 .+-. 0.69 AU 10.23
.+-. 0.51 AU 7.54 .+-. 0.00 AU Test products AU % AU % AU %
Hydroquinone 40.59 .+-. 2.41 -9 10.19 .+-. 1.21 0 7.73 .+-. 0.03 +3
formulated at 0.1% (w/v) Arbutin formulated 40.48 .+-. 0.85 +2
10.02 .+-. 0.13 -2 7.64 .+-. 0.19 +1 at 1% (w/v) Arbutin formulated
41.13 .+-. 0.54 +1 9.86 .+-. 0.18 -4 7.73 .+-. 0.34 +3 at 3% (w/v)
Kojic acid 40.11 .+-. 1.41 +3 10.90 .+-. 0.51 +7 7.67 .+-. 0.03 +3
formulated at 1% (w/v) Kojic acid 41.69 .+-. 0.65 0 9.36 .+-. 1.02
-8 7.13 .+-. 0.09 -5 formulated at 3% (w/v) N-Undecylenoyl- 41.34
.+-. 1.15 0 9.01 .+-. 1.33 -12 5.96 .+-. 0.42 -21 phenylalanine
formulated at 1% (w/v) N-Undecylenoyl- 45.228 .+-. 1.49 +9 8.78
.+-. 0.52 -14 5.38 .+-. 0.06 -29 phenylalanine formulated at 3%
(w/v)
[0162] 6.2--Measurement of the Intracellular Melanin Content
[0163] Three days after the final topical application and after the
chromametric measurement, the intracellular melanin is extracted
from the epidermides by incubation for 45 minutes at 100.degree. C.
in Soluene.TM. 350 (200 .mu.l/epidermis), as described in "Ozeki
H., Ito S., Wakamatsu K., Hirobe T.; J. Invest. Dermatol., 105,
1995, 361-366. The samples are centrifuged for 10 minutes at 10 000
rpm.
[0164] The extracted intracellular melamin is measured by
spectrophotometry at 500 nm. A melanin calibration range is
prepared in parallel.
[0165] The results are expressed as mg/ml of intracellular melanin
and as a percentage of inhibition relative to the control
group.
11 Inhibition of the amount of Intracellular melanin intracellular
Test products (control: 347 .+-. 2 .mu.g/ml) melanin Hydroquinone
317 .+-. 0 .mu.g/ml -9% formulated at 0.1% (w/v) Arbutin 242 .+-.
46 .mu.g/ml -28% formulated at 1% (w/v) Arbutin 263 .+-. 16
.mu.g/ml -24% formulated at 3% (w/v) Kojic acid 273 .+-. 2 .mu.g/ml
-21% formulated at 1% (w/v) Kojic acid 234 .+-. 19 .mu.g/ml -33%
formulated at 3% (w/v) N-Undecylenoylphenylalanine 264 .+-. 9
.mu.g/ml -24% formulated at 1% (w/v) N-Undecylenoylphenylalanine
264 .+-. 11 .mu.g/ml -24% formulated at 3% (w/v)
[0166] 6.3--Results Analysis
[0167] Hydroquinone, tested in topical application at a
concentration of 0.1% (w/v) in an emulsion, has no significant
effect either on the chromametric parameters L*, a* and b* or on
the melanin content of the reconstructed human epidermides. The
absence of a depigmenting effect of hydroquinone is due either to
the low test concentration, which was deliberately selected as
noncytotoxic, or to the short duration of the treatment.
[0168] Arbutin, tested in topical application at 1% and 3% (w/v) in
an emulsion, has no significant effect on the chromametric
parameters L*, a* and b*. However, it inhibits the melanin content
of the reconstructed human epidermides by 28% and 24%,
respectively.
[0169] Kojic acid, tested in topical application at 1% and 3% (w/v)
in an emulsion, has no significant effect on the chromametric
parameters L*, a* and b*. However, it inhibits the melanin content
of the reconstructed human epidermides by 21% and 33%,
respectively.
[0170] N-Undecylenoylphenylalanine, tested in topical application
at 1% (w/v) in an emulsion, inhibits the b* color parameter by 15%
and the melanin content of the reconstructed human epidermides by
24%.
[0171] At a concentration of 3% (w/v), N-undecylenoylphenylalanine
increases the L* parameter by 9% and concomitantly reduces the a*
color parameter by 14%, the b* color parameter by 29% and the
melanin content of the reconstructed epidermides by 24%.
[0172] These tests on reconstructed epidermides show that
N-Undecylenoylphenylalanine has improved depigmenting activity
compared with that of the reference products, insofar as it has an
influence both on the chromametric parameters and on the
intracellular melanin contents.
[0173] 7--Conclusion
[0174] The results obtained in this study together demonstrate
strong depigmenting activity of N-undecylenoylphenylalanine. This
activity is quantified both in melanocyte cultures and in a 3D
model composed of reconstructed human epidermides. In contrast to
the reference products, the depigmenting activity of
N-undecylenoylphenylalanine involves the MC1R receptors.
N-Undecylenoylphenylalanine is an MC1R receptor antagonist and
inhibits all the steps of the .alpha.-MSH cycle involved in
melanogenesis.
COSMETIC FORMULATION EXAMPLES
EXAMPLE 2
Lightening Care Emulsion for Mature Skin
[0175]
12 Montanov .TM. 202 02.00% Montanov .TM. 68 02.00% Caprylic capric
triglycerides 10.00% Squalane 10.00% Water q.s. 100%
N-Undecylenoylphenylalanine 01.00% Sepigel .TM. 305 00.70%
Magnesium ascorbyl phosphate 02.00% Sepicide .TM. HB 00.30%
Sepicide .TM. CI 00.20% Fragrance 00.50%
EXAMPLE 3
Lightening Firming Care Emulsion
[0176]
13 Montanov .TM. 202 03.00% 24% sodium hydroxide 00.06% Ethylhexyl
methoxycinnamate 06.00% Lanol .TM. 1688 08.00% Benzophenone-3
04.00% Water q.s. 100% N-Undecylenoylphenylalanine 02.00% Simulgel
.TM. NS 00.50% Sepilift .TM. DPHP 00.50% Dimethicone 02.00%
Cyclomethicone 02.00% Arbutin 0.3% Sepicide .TM. HB 00.30% Sepicide
.TM. CI 00.20% Fragrance 00.10%
EXAMPLE 4
Lightening Cream-Gel Containing .alpha.-Hydroxy Acids
[0177]
14 Hydroxyethylcellulose 00.80% Ethylhexyl octanoate 05.00% 60%
sodium lactate 14.00% Water q.s. 100% N-Undecylenoylphenylalanine
03.00% Sepigel .TM. 305 04.20% Sepicide .TM. HB 02.00% Sepicide
.TM. CI 03.00% Fragrance 00.10%
EXAMPLE 5
Lightening Care Emulsion
[0178]
15 Montanov .TM. L 01.00% Cetyl alcohol 02.00% Isodecyl
neopentanoate 12.00% Cetaryl octanoate 10.00% Glycerol 03.00% Water
q.s. 100% N-Undecylenoylphenylalanine 01.00% Simugel .TM. EG 02.00%
Kojic acid 01.00% Sepicide .TM. HB 00.30% Sepicide .TM. CI 00.20%
Fragrance 00.10%
EXAMPLE 6
Lightening Lotion
[0179]
16 Oramix .TM. CG110 05.00% Kathon .TM. CG 00.08% Water q.s. 100%
N-Undecylenoylphenylalanine 01.00% Fragrance 00.10%
[0180] This lotion may be sold in bottles or impregnated into
wipes.
[0181] The definitions of the commercial products used in the
examples are as follows:
[0182] Sepilift.TM. DHP (INCI name: dipalmitoyl hydroxyproline),
sold by the company SEPPIC.
[0183] Sepicide.TM. HB is a preserving mixture comprising
phenoxyethanol, methyl paraben, ethyl paraben, propyl paraben and
butyl paraben, sold by the company SEPPIC.
[0184] Sepicide.TM. CI is imidazolidinylurea, sold by the company
SEPPIC.
[0185] Sepicalm.TM. VG (INCI name: sodium palmitoyl proline and
extract of water lily flower), sold by the company SEPPIC.
[0186] Kathon.TM. CG (INCI name:
methylisothiazolinone/methylchloroisothia- zolinone).
[0187] Simulgel.TM. EG is a copolymer inverse latex (INCI name:
sodium acrylate/sodium acryloyldimethyltaurate copolymer and
isohexadecane and Polysorbate 80) sold by the company SEPPIC.
[0188] Simulgel.TM. NS is a copolymer inverse latex (INCI name:
hydroxyethyl acrylate/sodium acryloyldimethyltaurate copolymer and
squalane and Polysorbate 60) sold by the company SEPPIC.
[0189] Lanol.TM. 1688 is cetearyl ethylhexanoate, sold by the
company SEPPIC.
[0190] Sepigel.TM. 305 is a polymer inverse latex (INCI name:
polyacrylamide and C13-C14 isoparaffin and Laureth 7).
[0191] Montanov.TM. L is an emulsifier based on C14-C22 alcohol and
on C12-C20 alkyl polyglucoside.
[0192] Montanov.TM. 68 is an emulsifier based on cetearyl alcohol
and cetearyl polyglucoside.
[0193] Montanov.TM. 202 is an emulsifier based on arachidyl
alcohol, behenyl alcohol and arachidyl polyglucoside.
* * * * *