U.S. patent application number 11/001085 was filed with the patent office on 2005-10-06 for topical administration of at least one double-stranded rna oligonucleotide (dsrna).
This patent application is currently assigned to L'OREAL. Invention is credited to Collin-Djangone, Christine, Duranton, Albert, Pruche, Francis, Simonnet, Jean-Thierry.
Application Number | 20050222071 11/001085 |
Document ID | / |
Family ID | 29558920 |
Filed Date | 2005-10-06 |
United States Patent
Application |
20050222071 |
Kind Code |
A1 |
Duranton, Albert ; et
al. |
October 6, 2005 |
Topical administration of at least one double-stranded RNA
oligonucleotide (dsRNA)
Abstract
Expression of a messenger RNA which encodes a protein which is
expressed by differentiated cells, notably the cells of the skin or
appendages thereof, is inhibited by topically administering to said
differentiated cells a thus effective amount of at least one
double-stranded RNA oligonucleotide, formulated into a topically
applicable, physiologically acceptable medium therefor; such regime
or regimen is useful for treating a variety of afflictions or
conditions, e.g., combating the signs of skin aging, stimulating
hair growth or retarding loss thereof, inhibiting cellular
proliferation and/or differentiation, etc.
Inventors: |
Duranton, Albert; (Maison
Laffite, FR) ; Collin-Djangone, Christine;
(Amblainville, FR) ; Pruche, Francis; (Senlis,
FR) ; Simonnet, Jean-Thierry; (Paris, FR) |
Correspondence
Address: |
BUCHANAN INGERSOLL PC
(INCLUDING BURNS, DOANE, SWECKER & MATHIS)
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Assignee: |
L'OREAL
PARIS
FR
|
Family ID: |
29558920 |
Appl. No.: |
11/001085 |
Filed: |
December 2, 2004 |
Current U.S.
Class: |
514/44A ;
435/455 |
Current CPC
Class: |
A61K 8/606 20130101;
A61Q 19/08 20130101; A61Q 19/02 20130101; A61Q 19/007 20130101;
C12N 2310/13 20130101; A61K 31/7088 20130101; A61Q 7/00 20130101;
A61Q 19/00 20130101 |
Class at
Publication: |
514/044 ;
435/455 |
International
Class: |
A61K 048/00; C12N
015/85 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 3, 2002 |
FR |
02/06796 |
Jun 2, 2003 |
WO |
PCT/FR03/01648 |
Claims
What is claimed is:
1. A regime or regimen for inhibiting the expression of a messenger
RNA which encodes a protein which is expressed by differentiated
cells, comprising topically administering to said differentiated
cells a thus effective amount of at least one double-stranded RNA
oligonucleotide, formulated into a topically applicable,
physiologically acceptable medium therefor.
2. A regime or regimen for inhibiting the expression of a messenger
RNA which encodes a protein which is expressed by differentiated
cells of the skin or appendages thereof, comprising topically
administering to said differentiated cells of the skin or
appendages thereof a thus effective amount of at least one
double-stranded RNA oligonucleotide, formulated into a topically
applicable, physiologically acceptable medium therefor.
3. The regime or regimen as defined by claim 2, said at least one
double-stranded RNA oligonucleotide comprising at least 10
nucleotides.
4. The regime or regimen as defined by claim 2, said at least one
double-stranded RNA oligonucleotide comprising from 12 to 40
nucleotides.
5. The regime or regimen as defined by claim 2, said at least one
double-stranded RNA oligonucleotide comprising from 20 to 25
nucleotides.
6. The regime or regimen as defined by claim 2, said at least one
double-stranded RNA oligonucleotide comprising from 21 to 23
nucleotides.
7. The regime or regimen as defined by claim 2, said at least one
double-stranded RNA oligonucleotide having been obtained by the
folding and pairing of a self-complementary RNA single strand.
8. The regime or regimen as defined by claim 2, said at least one
double-stranded RNA oligonucleotide having unpaired ends of from 2
to 6 nucleotides in length.
9. A regime or regimen as defined by claim 2 for depigmenting
and/or bleaching the skin or the hair and nails of an individual in
need of such treatment, said at least one double-stranded RNA
oligonucleotide inhibiting the expression of a messenger RNA which
encodes tyrosinase and/or TRP-1.
10. A regime or regimen as defined by claim 2 for preventing and/or
combating cutaneous signs of aging of an individual in need of such
treatment, said at least one double-stranded RNA oligonucleotide
inhibiting the expression of a messenger RNA which encodes
neutrophil elastase and/or hyaluronidase and/or a metalloproteinase
and/or HMG-CoA reductase and/or calmodulin-like skin protein (CLSP)
and/or NO synthase.
11. A regime or regimen as defined by claim 2 for inducing and/or
stimulating growth of head hair and/or body hair and/or retarding
loss of head hair of an individual in need of such treatment, said
at least one double-stranded RNA oligonucleotide inhibiting the
expression of a messenger RNA which encodes a metalloprotease
and/or type I or type II 5.alpha.-reductase and/or NO synthase.
12. A regime or regimen as defined by claim 2 for preventing and/or
combating the loss of firmness and/or suppleness of the skin and/or
atropy of the skin and/or the formation of wrinkles and fine lines
of an individual in need of such treatment, said at least one
double-stranded RNA oligonucleotide inhibiting the expression of a
messenger RNA which encodes the enzyme neutrophil elastase,
hyaluronidase or a metalloprotease.
13. A regime or regimen as defined by claim 2 for preventing and/or
combating excessive production of sebum or sweat in the region of
the armpits or the feet of an individual in need of such treatment,
said at least one double-stranded RNA oligonucleotide inhibiting
the expression of a messenger RNA which encodes HMG-COA
reductase.
14. A regime or regimen as defined by claim 2 for treating an
androgen-dependent disorder of an individual in need of such
treatment, said at least one double-stranded RNA oligonucleotide
inhibiting the expression of a messenger RNA which encodes type I
.alpha.-reductase.
15. A regime or regimen as defined by claim 2 for treating
hyperseborrhea and/or acne of an individual in need of such
treatment, said at least one double-stranded RNA oligonucleotide
inhibiting the expression of a messenger RNA which encodes type I
.alpha.-reductase.
16. A regime or regimen as defined by claim 2 for combating the
harmful effects of ultraviolet radiation and/or treating dry skin
of an individual in need of such treatment, said at least one
double-stranded RNA oligonucleotide inhibiting the expression of a
messenger RNA which encodes calmodulin-like skin protein
(CLSP).
17. A regime or regimen as defined by claim 2 for inhibiting
cellular proliferation and/or differentiation and/or inhibiting the
breakdown and/or destruction of the cells of the skin of an
individual in need of such treatment, said at least one
double-stranded RNA oligonucleotide inhibiting the expression of a
messenger RNA which encodes NO synthase.
18. A regime or regimen as defined by claim 2 for treating
sensitive skin and erythemas of an individual in need of such
treatment, said at least one double-stranded RNA oligonucleotide
inhibiting the expression of a messenger RNA which encodes NO
synthase.
19. A regime or regimen for inhibiting the expression of a
messenger RNA which encodes a protein which is expressed by
differentiated cells of a eukaryotic microorganism on the surface
of the skin or appendages thereof, comprising topically
administering to said differentiated cells of said eukaryotic
microorganism a thus effective amount of at least one
double-stranded RNA oligonucleotide, formulated into a topically
applicable, physiologically acceptable medium therefor.
20. A regime or regimen as defined by claim 2 for inhibiting the
expression of a messenger RNA which encodes a protein expressed by
melanocytes and/or keratinocytes and/or fibroblasts and/or
endothelial cells and/or resident immune cells.
21. A regime or regimen as defined by claim 2 for inhibiting the
expression of a messenger RNA which encodes a protein selected from
the group consisting of tyrosinase, TRP-1 (tyrosinase-related
protein 1), the enzyme neutophil elastase, hyaluronidase, a
metalloproteinase, HMG-CoA reductase, type I or type II
5.alpha.-reductase, calmodulin-like skin protein (CLSP), NO
synthase, urokiinase, cyclooxygenases, lipoxygenases,
phospholipases, 15-PGDH, enzymes of hormonal metabolism, type I or
type II 5.alpha.-reductase, matrix proteins of the elastin or
collagen type, keratinocyte differentiation proteins of the
cytokeratin type, proteins involved in moisturization of the skin,
filaggrin, aquaporins, a serine protease, a cytokine, a growth
factor, a protein or a peptide of morphogenesis, proteins involved
in the antibacterial defenses of the skin, hBD2, hBD3, dermcidin,
RNase 7.
22. A regime or regimen as defined by claim 2 for inhibiting the
expression of a messenger RNA which encodes a protein of a
eukaryotic microorganism selected from the group consisting of
squalene epoxidase, C. albicans cytochrome P450-dependent 14-alpha
sterol demethylase, proteins for detoxifying antimycotic agents,
trichophyton rubrum esterases, aspartic proteinases, phospholipase
D, Candida phospholipase B, 2,3-oxidosqualene cyclases,
22,23-epoxy-2-aza-2,3-dihydrosqualene (EAS) and azasqualene alcohol
(ASA).
23. A regime or regimen as defined by claim 2 for treating
psoriasis of an individual in need of such treatment, said at least
one double-stranded RNA oligonucleotide inhibiting the expression
of a messenger RNA which encodes a metalloprotease and/or a
detoxification protein.
24. A topically applicable cosmetic/dermatological composition,
comprising an amount of at least one double-stranded RNA
oligonucleotide effective, when administered to an individual in
need of such treatment, for inhibiting the expression of a
messenger RNA which encodes a protein which is expressed by
differentiated cells, formulated into a topically applicable,
physiologically acceptable medium therefor.
25. The topically applicable cosmetic/dermatological composition as
defined by claim 24, said at least one double-stranded RNA
oligonucleotide comprising from 5.10.sup.-7 to 5% by weight
thereof.
26. The topically applicable cosmetic/dermatological composition as
defined by claim 24, said at least one double-stranded RNA
oligonucleotide comprising a complex thereof.
27. The topically applicable cosmetic/dermatological composition as
defined by claim 24, said at least one double-stranded RNA
oligonucleotide being confined in the core or a wall of a delivery
vehicle therefor.
28. The topically applicable cosmetic/dermatological composition as
defined by claim 27, said at least one double-stranded RNA
oligonucleotide being encapsulated in the core or the wall of
microspheres, nanospheres, oleosomes, niosomes or nanocapsules.
29. The topically applicable cosmetic/dermatological composition as
defined by claim 24, formulated as an aqueous or oily solution,
emulsion, gel, paste, solid, cream, ointment, milk, lotion, serum,
mousse or aerosol.
30. The topically applicable cosmetic/dermatological composition as
defined by claim 24, confined on a microneedle patch.
31. The topically applicable cosmetic/dermatological composition as
defined by claim 24, said at least one double-stranded RNA
oligonucleotide comprising two of the sequences selected from the
group consisting of:
5 5'-UGCACCACUUGGGCCUCAAdTdT,: SEQ ID no.1
5'-UUGAGGCCCAAGUGGUGCAdTdT,: SEQ ID no.2
5'-CGGCUACGACCCCGUAAACdTdT,: SEQ ID no.3
5'-GUUUACGGGGUCGUAGCCGdTdT,: SEQ ID no.4
5'-AGCCGCGAGGGUCGUCCAAdTdT,: SEQ ID no.5
5'-UUGGACGACCCUCGCGGCUdTdT,: SEQ ID no.6
5'-CUGCAUCCUCCUGGCCAUGdTdT,: SEQ ID no.7
5'-CAUGGCCAGGAGGAUGCAGdTdT,: SEQ ID no.8
5'-GGCUUUCUCCGCGGUUGACdTdT,: SEQ ID no.9
5'-GUCAACCGCGGAGAAAGCCdTdT,: SEQ ID no.10
5'-AAGUGUUUGAUGCUGGAGGdTdT,: SEQ ID no.11
5'-CCUCCAGCAUCAAACACUUdTdT,: SEQ ID no.12
5'-GCACCAGCUUUUCUGCCUUdTdT,: SEQ ID no.13
5'-AAGGCAGAAAAGCUGGUGCdTdT,: SEQ ID no.14
5'-ACUGCACAGAGAGACGACUdTdT,: SEQ ID no.15
5'-AGUCGUCUCUCUGUGCAGUdTdT,: SEQ ID no.16
5'-GCACCAGCUUUUCUGCCUUdTdT,: SEQ ID no.17
5'-AAGGCAGAAAAGCUGGUGCdTdT,: SEQ ID no.18
5'-AGCAGCAUGCACAAUGCCUdTdT,: SEQ ID no.19
5'-AGGCAUUGUGCAUGCUGCUdTdT,: SEQ ID no.20
5'-AGCCUGACCUCACUCUAACdTdT,: SEQ ID no.21 and
5'-GUUAGAGUGAGGUCAGGCUdTdT,: SEQ ID no.22
Description
CROSS-REFERENCE TO PRIORITY/PCT/PROVISIONAL APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn. 119
of FR-02/06796, filed Jun. 3, 2002, and of provisional application
Ser. No. 60/386,720, filed Jun. 10, 2002, and is a continuation of
PCT/FR 2003/001648, filed Jun. 2, 2003 and designating the United
States (published in the French language on Dec. 11, 2003, as WO
2003/101376 A3; the title and abstract were also published in
English), each hereby expressly incorporated by reference and each
assigned to the assignee hereof.
BACKGROUND OF THE INVENTION
[0002] 1. Technical Field of the Invention
[0003] The present invention relates to the administration of at
least one double-stranded RNA oligonucleotide (dsRNA) via topical
application and to compositions for topical administration which
comprise at least one encapsulated double-stranded RNA
oligonucleotide formulated into a physiologically acceptable medium
therefor.
[0004] 2. Description of Background and/or Related and/or Prior
Art
[0005] A large number of substances are administered topically for
compensating for or preventing certain aesthetic impairments or
certain disorders of the skin or its appendages. Of these
substances, those which exhibit true biological specificity are
more particularly desired.
[0006] Thus, the greater the extent to which the substances exhibit
a specific activity, such as a specific biological activity, the
less likely it is that they will induce undesirable side effects.
For example, preference will be given to formulating a substance
whose action is targeted on an enzyme such as tyrosinase into a
composition which seeks to clear the complexion or prevent
undesirable pigmentation. Mention may also be made, by way of
example, of formulating a substance which acts specifically on
certain metalloproteinases or elastases into a composition for
preventing aging.
[0007] To date, this problem of specificity has been resolved by
administering native DNA or RNA oligonucleotides or antisense DNA.
However, such application has its limitations.
[0008] Thus, including DNA, non-specific RNA or antisense DNA
oligonucleotides (such as those described, for example, in WO
01/58918) in topical formulations has not enabled any desirable
efficacy to be obtained. In particular, RNases on the surface of
the skin degrade native RNA and it is not possible for the
quantities reaching the target mRNAs to be sufficient to obtain the
desired effect. Another limitation to administering single-stranded
oligonucleotides such as those mentioned above is the risk of the
oligonucleotide possibly undergoing secondary refolding (or
secondary re-pairing). For this reason, administering
single-stranded oligonucleotides (such as antisense DNA
oligonucleotides) in vivo is frequently ineffective. Furthermore,
the stability of the effects produced by single-stranded
oligonucleotides in vivo is short because of the oligonucleotides
being broken down rapidly intracellularly and having a short
extracellular half-life in vivo (Khan A et al., J. Drug Target,
2000, 8, 319-334).
[0009] Thus a need continues to exist for a composition which
resolves both the difficulties of administering active compounds in
a targeted manner and the problem of specificity and which is
suitable for topical application.
[0010] Double-stranded RNA oligonucleotides, also termed dsRNAs,
have long been known to be present in plants and eukaryotic
organisms (Haines D. S. et al., J. Cell Biochem., 1991; 46:9-20);
their role is to inhibit the expression of a specific gene (Fire
A., Trends in Genetic, 1999; 15:358-363).
[0011] Using dsRNA for therapeutic purposes has been described in
WO 01/36646, which proposes a method for inhibiting genes which are
involved in diseases, in particular cancers. The examples of
implementing this method propose inhibiting the expression of a
gene in undifferentiated cells, such as embryonic cells, or
oocytes, by intracellularly injecting dsRNA which is targeted at
the gene to be inhibited. The objective is to obtain a systemic
effect. The modes of administration envisaged concern general
administrations.
[0012] Contrary to the pharmaceutical compositions which are
proposed in this publication, a topical formulation should reach a
specific differentiated cell while at the same time only acting at
the superficial level, that is to say at the level of the skin or
its appendages (dermis, epidermis and keratinized appendages), or
even only at the surface of the skin by targeting the eukaryotic
microorganisms which cover it. It is also possible to search for an
effect on a specific skin cell type, such as a fibroblast, a
melanocyte, a keratinocyte, a Langerhans cell or an endothelial
cell which is involved in aesthetic impairments or particular
dermatological disorders.
SUMMARY OF THE INVENTION
[0013] It has now surprisingly been found that double-stranded RNA
oligonucleotides can be administered for specifically inhibiting
the expression of certain proteins in differentiated cells, in
particular skin cells, and therefore for formulating compositions
suitable for external topical administration.
[0014] The present invention thus features topically administering
at least one double-stranded RNA oligonucleotide which is active on
differentiated cells such as the cells of the dermis and the
epidermis (melanocytes, fibroblasts, keratinocytes, etc.).
[0015] Topical administration according to the invention both
possesses the features mentioned above and makes it possible to
ameliorate or overcome the difficulties which have thus far been
encountered.
[0016] This invention specifically features the expression of
proteins in the cells of the dermis and the epidermis or in
eukaryotic microorganisms at the surface of the skin, with it being
possible for the targeting of the cells to be effected, in
particular, by selecting a coating which enables the
double-stranded RNA oligonucleotides to spread in the cell which is
targeted.
[0017] The double-stranded RNA oligonucleotides make it possible to
specifically inhibit a gene (or several genes in the case of a
composition which combines double-stranded RNA oligonucleotides
which possess different sequences and which are targeted at mRNAs
encoding different proteins).
[0018] Because of their duplex structure, the double-stranded RNA
oligonucleotides are resistant to RNases (enzymes for degrading
RNA); the oligonucleotides are not broken down at the surface of
the skin.
[0019] Too, because of their duplex structure, these
double-stranded RNA oligonucleotides are unable to undergo folding
and self-pairing; in this way, the bioavailability of the active
product, that is to say the quantity of double-stranded RNA
oligonucleotide which produces the desired effect and which arises
in the cells, is markedly improved and makes it possible to use
lower quantities of double-stranded RNA oligonucleotide.
[0020] The compositions comprising the double-stranded RNA
according to the invention are particularly suitable for cosmetic
or dermatological administration since they make it possible to
reach the target cell without an invasive mode of administration
and are active without having to penetrate into the cell
nucleus.
DETAILED DESCRIPTION OF BEST MODE AND SPECIFIC/PREFERRED
EMBODIMENTS OF THE INVENTION
[0021] The cell machinery which induces inhibition of the
expression of the corresponding mRNA is brought into play by means
of a complex mechanism within the cytoplasm. Administering a
double-stranded RNA oligonucleotide into a cell induces a
phenomenon involving the post-transcriptional extinction of the
mRNA which the oligonucleotide targets.
[0022] The molecular mechanism which is brought into play involves
sequences of 21 to 23 nucleotides which are contained in the
double-stranded RNA oligonucleotides and which are responsible for
the specificity for the sequence of the mRNA. These double-stranded
RNA oligonucleotides are also termed dsRNA or else siRNA (short
interfering RNA, see Tuschl. T., Chem. Biochem., 2001; 2:239-245).
The mechanism involves a dsRNA-protein complex which is formed in
an ATP-dependent manner and which initiates a reaction in which the
targeted mRNA is broken down specifically (Nykanen A et al., Cell,
2001, 107, 309-321).
[0023] The present invention firstly features topical
administration/application of at least one double-stranded RNA
oligonucleotide which generally comprises at least 10 nucleotides,
in particular from 12 to 40 nucleotides, preferably from 20 to 25
nucleotides; very particular preference is given to double-stranded
RNA oligonucleotides which comprise from 21 to 23 nucleotides.
[0024] A "double-stranded RNA oligonucleotide" is understood as
being a ribonucleic acid sequence which has a double-helical
structure whose sequence is substantially identical to at least a
part of the targeted messenger RNA.
[0025] These sequences of the double-stranded RNA oligonucleotides
according to the invention are generally derived from endogenous
sequences, that is to say they represent all or part of mammalian
or eukaryotic microorganism nucleotide sequences. The sequence can
be a gene sequence or a coding DNA (cDNA) sequence which is
produced from mammalian-derived messenger RNA (mRNA) using reverse
transcriptase, for example; the sequence can also be a sequence of
a yeast gene.
[0026] In particular, when the double-stranded RNA oligonucleotide
sequence corresponds to all or part of the sequence of a gene,
preference is given to using the sequences of one or more exons of
the gene concerned.
[0027] The double-stranded RNA oligonucleotides according to the
invention can be double-stranded RNA oligonucleotides which
comprise one or more nucleotides which have been modified by
substitution, deletion or insertion, with these modifications being
such that the sequence of the double-stranded RNA oligonucleotide
enables the oligonucleotide to specifically recognize a fragment of
the mRNA which is the target of the degradation mechanism.
[0028] The double-stranded RNA oligonucleotides may also have a
modified skeleton which improves their stability, for example.
[0029] For example, the phosphodiester bonds of the natural RNA
strands can be modified so as to include at least one nitrogen atom
or sulfur atom. Furthermore, the double-stranded RNA
oligonucleotides according to the invention can contain bases other
than the 4 usual bases.
[0030] The double-stranded structure of the double-stranded RNA
oligonucleotide can be obtained by pairing two single RNA strands
which are complementary or else the double-stranded RNA
oligonucleotide can be obtained by the folding and pairing of a
unique "self-complementary" single RNA strand, that is to say a
strand which comprises two fragments of complementary sequence
which can pair by the single strand being folded to form a double
helix.
[0031] "Double-stranded RNA oligonucleotide which is substantially
identical to a gene fragment" is understood as meaning a
double-stranded RNA oligonucleotide whose sequence possesses a
degree of homology (percentage of nucleic acid bases which are
identical between two sequences, see the calculation methods
proposed by Atschul et al., J. Molec. Biol., 1990, 215:403) with
the fragment of said gene which is between 80 and 100% and
preferably at least 90%.
[0032] In one preferred embodiment of the invention, the
double-stranded RNA oligonucleotide exhibits unpaired ends of 2 to
6 nucleotides in length.
[0033] The double-stranded RNA oligonucleotide according to the
invention can also be modified by the addition of a polyethylene
glycol, as defined by Garrett et al. (Bioorg. Med. Chem., 2000
July, 8(7): 19779-97), in order to improve the efficacy of its
transfection into the host cell.
[0034] The double-stranded RNA oligonucleotides according to the
invention can be synthesized manually or automatically using a
large number of in vivo or in vitro synthesis methods.
[0035] The in vitro synthesis methods can be chemical or enzymic,
for example using an RNA polymerase (T3, T7 or SP6 by way of
example) which will transcribe a chosen DNA (or cDNA) sequence
model.
[0036] A large number of techniques for synthesizing
double-stranded RNA in vivo are described in the literature; they
can be carried out in a variety of bacterial or higher organism
cell types (Sambrook et al., Molecular Cloning, A Laboratory
Manual, Second Edition (1989), DNA cloning, volume 1 and 11, D. N.
Glover (ed. 1985), Oligonucleotide Synthesis, M. J. Gaits (ed.
1984), Nucleic Acid Hybridation, B. D. Hames and S. J. Higgins (ed.
1984), Transcription and Translation, B. D. Hames and S. J. Higgins
(ed. 1984), Animal Cell Culture, R. I. Freshney (ed. 1986),
Immobilised Cells and Enzymes, IRL Press (1986), B. Pertal, A
Practical Guide to Molecular Cloning, (1984), Gene Transfer Vectors
for Mammalian Cells, J. H. Miller and M. P. Calos, Cold Spring
Harbor Laboratory (ed. 1987), Methods of Enzymology, vol. 154, Wu
and Grossman, and 155, Wu, Mayer and Walker (1987), Immunochemical
Methods in Cell and Molecular Biology, Academic Press, London,
Scopes (1987), Protein Purification: Principle and Practice,
2.sup.nd ed., Springer-Verlag, N.-Y. and Handbook of Experimental
Immunology, vol. I-IV, C. D. Weir and C. C. Blackwell (1986)). See
also the synthesis methods which are described in WO 01/36646 and
WO 01/75164.
[0037] The sequences of the double-stranded RNA oligonucleotides
which are employed are in each case selected, using any method of
molecular biology, for a specific topical use. While the mRNAs of
the biological targets which are of interest in the cosmetic or
dermatological field can, by way of example, be the proteins which
are described below, they can also be any mRNAs which encode other
skin proteins.
[0038] It is also possible to use a combination of several
double-stranded RNA oligonucleotides which possess different
sequences, and each of which has a different activity, for the
purpose, for example, of obtaining complementary or synergistic
effects.
[0039] In particular, the present invention relates to the topical
administration of at least one double-stranded RNA oligonucleotide
which is able to inhibit the expression of a messenger RNA which
encodes a protein which is expressed by a cell of the skin or of
its appendages.
[0040] The present invention also features cosmetic compositions in
which the double-stranded RNA oligonucleotide is able to inhibit
the expression of a messenger RNA which encodes a protein which is
expressed by a eukaryotic microorganism which is present at the
surface of the skin or of the scalp.
[0041] In particular, the skin protein is a protein which is
expressed by melanocytes and/or keratinocytes and/or fibroblasts
and/or the endothelial cells or the resident immune cells, such as
the Langerhans cells. The protein is preferably expressed by the
keratinocytes.
[0042] The proteins and/or oligopeptides whose synthesis can be
reduced or inhibited are those which are involved in the phenomena
of cell differentiation or proliferation, such as:
[0043] the growth factors EGF, TNF-.alpha., TGF, endothelin, NGF,
HGF, IGF and VEGF;
[0044] cytokines, for example of the IL1, IL6, IL8, etc. type;
[0045] receptors of the EGFr, TGFr, PAR, PPAR, FXR, RXR, CB1R,
CB2R, VR1, CRAB2, etc. type;
[0046] calcium-binding proteins of the calmodulin, CLP and CLSP
type, and those of the S100 protein family such as S100A8, S100A9,
S100A7, etc.;
[0047] calcineurin;
[0048] transglutaminases, for example transglutaminases 1, 3 or
5;
[0049] proteins ensuring intercellular cohesion/linkage, such as
occludins, laminins, caveolins, desmogleins, desmocollins,
corneodesmosins, plakoglobins, desmoplakins, etc.;
[0050] enzymes involved in post-translational modifications of
proteins such as phosphatases or protein phosphatases, for example
calcineurin, phosphorylases, protein kinases (e.g., PKC), glucosyl
transferases, peptidyl-arginine-deiminases, etc.;
[0051] proteases (MMP, for example 1, 2, 3 and 9, elastases,
aspartic acid proteases such as cathepsin-E and cathepsin-D,
cystein proteases of the cathepsin-L, B or H type, cathepsin L2,
SCCL, chymotrypsin equivalents, for example of the SCCE (kallicrein
7) type, trypsin-like, for example of the SCTE (kallicrein 5) type,
urokinase, SASPase, caspase, more specifically caspase 14,
calpains, proteases of the subtilisin-like proprotein convertase
type which are involved in the hydrolysis of filaggrin, such as
furin, PACE4, PC5/6 and PC7/8, proteases of the serine protease
family of the transmembrane type, for example matriptase and/or
their endogenous inhibitors such as TIMP, PAIl, PAI2,
antileukoprotease, elafin, LEKTI, cystatin A, cystatin M/E,
etc.;
[0052] exoglycosidases and endoglycosidases, for example of the
heparanase type, hyaluronidases, chondroitinases, aspartyl
glucosaminidase, B glycosidase, a glycosidases, etc., and their
endogenous inhibitors;
[0053] enzymes of lipid metabolism, such as HMGCoA reductase,
cholesterol sulfatases or sulfotransferases, sphingomyelinases,
ceramidases, etc.;
[0054] tyrosinase, TRP-1 or TRP-2;
[0055] enzymes of eicosanoid metabolism, such as cyclooxygenases,
lipoxygenases, phospholipases, 15-PGDH, etc.;
[0056] enzymes of hormonal metabolism, such as type I or type II
5.alpha.-reductase;
[0057] matrix proteins of the elastin, collagen, etc. type;
[0058] keratinocyte differentiation proteins of the cytokeratin
type;
[0059] proteins involved in hydration of the skin such as
filaggrin, aquaporins, etc.;
[0060] proteins involved in the antibacterial defenses of the skin,
hBD2, hBD3, dermcidin, RNase 7, etc.;
[0061] Other nonlimiting examples of proteins or peptides whose
expression and/or activity is intended to be inhibited are reported
in Textbook of Dermatology, eds. R H Champion, J L Burton, D A
Burns, S M Breathnach, sixth edition, 1998, Blackwell Science Ltd
ISBN 0-632-03796-2.
[0062] The messenger RNA in the dermis whose expression can be
reduced or inhibited can be that of the fibroblasts or of other
cells which are present in the dermis, i.e., the cells of the blood
vessels, the cells of the epidermal appendages (examples:
sebocytes, sudoriparous glands, etc.) and the cells which are able
to migrate into the dermis such as the cells which are involved in
immunity or inflammation.
[0063] The proteins and/or oligopeptides whose synthesis can be
reduced or inhibited are those which are involved in the phenomena
of cell differentiation or proliferation, such as those described
in the case of keratinocytes and, in addition:
[0064] the growth factors EGF, TGF, endothelin, NGF, HGF and FGF,
etc.;
[0065] phosphatases;
[0066] transglutaminases;
[0067] phosphorylases;
[0068] proteins involved in the renewal of the extracellular matrix
(proteases: MMP metalloproteases, serine proteases such as
urokinase, tPA, hyaluronidases);
[0069] structural proteins of the dermis such as collagen, or of
the amorphous ground substance such as the proteoglycan
proteins;
[0070] proteins involved in the maturation of the dermis such as
lysyl oxidase and lysyl hydroxylase.
[0071] At the level of complex structures, such as the hair
follicle, it is envisaged to administer a group of siRNAs, each of
which is directed against a messenger which is known to encode a
regulatory and/or structural protein of the hair shaft, in order to
obtain the desired effect:
[0072] antigrowth: for example the proteins involved in the cell
cycle, and/or IGF receptor and/or T4 thyroid receptor;
[0073] antiloss: for example the siRNAs which are directed against
cytokines IL1, IL6, TNFalpha and MCP1 and/or MMP proteases,
urokinase and/or lipoxygenase;
[0074] regrowth: for example the proteins involved in breaking down
PGF2alpha and/or 5alpha reductase; proteins involved in
morphogenesis such as alpha3beta1-integrin, betacatenin, laminin-10
and LEF-1;
[0075] shaping the hair shaft, such as curling or uncurling: for
example, the proteins involved in differentiation of the hair
shaft, such as acid or basic hair keratins and also the enzymes
associated with crosslinking the proteins of the shaft (e.g.,:
thiol oxidoreductases and transglutaminases 3 and 5).
[0076] Reference may be made to Jamora C., DasGupta R.,
Kocieniewski P., Fuchs E., Nature, 2003 Mar. 20;422(6929): 317-22
for a nonlimiting list of this type of proteins.
[0077] At the level of complex structures such as the sebaceous
gland, it is envisaged administering a group of siRNAs, each of
which is directed against proteins which are involved in synthesis
of the sebum, such as HMGCoa reductase and squalene synthase.
[0078] In the case of all these cell types, it is possible to
administer, according to the invention, siRNAs which are directed
against the expression of pathological proteins and/or
oligopeptides, in particular those linked to viral action (e.g.,
verrucal HPV) or to cancerous cells, or those which are
overexpressed in certain diseases. Mention may be made, for
example, of certain cytokines (for example IL1, TNFalpha-308 and
TNFbeta+252), receptor proteins (for example Toll-like receptors,
TLRs), certain proteins involved in proliferation such as
phosphatidylinositol 3 kinase, adhesion molecules (for example
CDw60), proteases, in particular serine proteases (stratum corneum
chimotrypsin enzyme, SCCE) or metalloproteases (in particular MMP-9
and MMP-19 involved in psoriasis), or else calcium-linked proteins
(calmodulin-like serin protease, CLSP).
[0079] In the case of all these cell types, it is also possible, in
accordance with the invention, to administer siRNAs which are
directed against the expression of proteins and/or oligopeptides
which are involved in the deactivation of a drug which is intended
for treating the skin, for example detoxification proteins such as
cytochrome P450 or cytochrome CYP2S1 in the case of psoriasis.
[0080] In the case of all these cell types, it is possible to
administer, in accordance with the invention, siRNAs which are
directed against the expression of proteins and/or oligopeptides
which are induced by external deleterious changes, for example
siRNAs which are directed against P2X purinergic receptor following
mechanical/chemical impairment of the corneal barrier.
[0081] The microorganisms which are present at its surface, and in
which it is possible to reduce or inhibit the synthesis of proteins
and/or oligopeptides, are eukaryotes, such as yeasts and fungi. In
a nonlimiting manner, representative are:
[0082] dermatophyte organisms which are the agents responsible for
mycoses (trichophyton species, including T. rubrum and T.
mentagrophytes). The dermatophytes are keratinophilic filamentous
fungi, that is to say having a preferential tropism for the hair
and nails and the corneal layer.
[0083] Three types of dermatophyte are responsible for
dermatophytoses:
[0084] 1. Anthropophilic dermatophytes: these are strictly of human
origin.
[0085] Examples of these agents are:
[0086] Trichophyton rubrum, Trichophyton interdigitale,
Trichophyton Violaceum, T. Rosaceeum, T. Tonsurans, T. Soudanensae,
Trichophyton Schoenleinii, Epidermophyton Floccosum Microsporum
Audouinii.
[0087] 2. Zoophilic dermatophytes which are transmitted to man by
animals. Examples of the agents responsible are Microsporum canis,
Trichophyton Mentagrophytes and Trichophyton Ochraceum.
[0088] 3. Geophilic dermatophytes, which are transmitted to man by
the soil. The main agent is microsporum gypseum.
[0089] Nondermatophytes (the Candida species such as C. albicans,
etc., the Scopulariopsis species such as S. brevicaulis, and
Malassezia spp.). These are yeasts represented by the genus candida
and by Malassezia furfur (formerly called pityrosporon). Candida
affects the skin, the nails and hair and the mucous membranes.
Malassezia furfur, which is a frequent saprophyte of the skin,
especially seborrheic skin, is the agent of pityriasis versicolor.
While it is entirely normal for particles of dead skin to become
detached from the scalp as from the remainder of the skin, this
permanent process of epidermal renewal can sometimes assume
embarrassing proportions. While being unsightly, dandruff is also
sometimes accompanied by more serious problems of the scalp such as
irritation, redness and itching. This is then referred to as
seborrheic dermatitis, a skin complaint which is promoted by the
fungus Pityrosporum ovale.
[0090] Molds, which are less frequently involved in conditions of
the corneal layer. They are responsible for certain onychomycoses
and invasive mycoses.
[0091] According to the invention, specific proteins of these
organisms such as C. albicans cytochrome P450-dependent
14-alphasterol demethylase and squalene epoxidase, proteins for
detoxifying antimycotic agents, such as the Trichophyton rubrum
esterases, aspartic proteinases, candida phospholipase B and
phospholipase D, and 2,3-oxidosqualene cyclases such as
22,23-epoxy-2-aza-2,3-dihydrosqualene (EAS) and azasqualene alcohol
(ASA) can be inhibited. A more complete list, by way of nonlimiting
examples, of eukaryotic organisms will be found in Textbook of
Dermatology, eds. R H Champion, J L Burton, D A Burns, S M
Breathnach, sixth edition, 1998, Blackwell Science Ltd, ISBN
0-632-03796-2.
[0092] More generally according to the invention, the siRNAs are
chosen and/or combined in accordance with the desired topical
effect. With the prior art, a molecular method can be considered
for streaming the messenger RNAs which are induced and/or repressed
by a drug or as the result of undesirable effects (DNA array
method) in order to determine the choice and the assembly of the
siRNAs which correspond to the effect to be obtained and/or
prevented.
[0093] The dsRNAs which are employed are in each case selected for
a specific topical use using any suitable molecular biological
method and such that this is done for each messenger RNA, in
particular in accordance with its primary structure. The mRNAs of
the biological targets which are of interest in cosmetics can, by
way of example, and with this being in a nonlimiting list, be:
tyrosinase, TRP-1, elastase, hyaluronidase, metalloprotease, HmoCoA
reductase, 5.alpha.-reductase, SCCE, NO synthase, urokinase, mRNAs
for epidermal proteins, etc.
[0094] In one embodiment of the invention, a double-stranded RNA
oligonucleotide is administered which is able to inhibit the
expression of a messenger RNA which encodes tyrosinase. In
particular, the double-stranded RNA oligonucleotide is such that
the sequence of its 5' sense strand is SEQ ID no. 1:
5'-UGCACCACUUGGGCCUCMdTdT and the sequence of its 5' antisense
strand is SEQ ID no. 2: 5'-UUGAGGCCCAAGUGGUGCAdTdT.
[0095] Administration according to the invention can also comprise
utilizing a double-stranded RNA oligonucleotide which is able to
inhibit the expression of a messenger RNA which encodes TRP-1
(tyrosinase-related protein 1).
[0096] Such topical applications are suitable for depigmenting
and/or bleaching the skin and/or the hair and nails.
[0097] Another embodiment of the invention employs a
double-stranded RNA oligonucleotide which is able to inhibit the
expression of a messenger RNA which encodes the enzyme neutrophil
elastase.
[0098] In particular, the double-stranded RNA oligonucleotide is
such that the sequence of its 5' sense strand is SEQ ID no. 3:
5'-CGGCUACGACCCCGUAAACdTdT and such that the sequence of its 5'
antisense strand is SEQ ID no. 4: 5'-GUUUACGGGGUCGUAGCCGdTdT.
[0099] Such a topical application is useful to prevent and/or
combat signs of skin aging such as the loss of firmness and/or
suppleness of the skin and/or atrophy of the skin and/or the
formation of wrinkles and fine lines.
[0100] Another embodiment of the invention uses a double-stranded
RNA oligonucleotide which is able to inhibit the expression of a
messenger RNA which encodes hyaluronidase.
[0101] Such a topical application is useful to prevent and/or
combat signs of skin aging such as loss of firmness and/or
suppleness of the skin and/or atrophy of the skin and/or the
formation of wrinkles and fine lines.
[0102] Another embodiment of the invention uses a double-stranded
RNA oligonucleotide which is able to inhibit the expression of a
messenger RNA which encodes a metalloproteinase.
[0103] The metalloproteinases (MMPs) are described, in particular,
in the article by Y. HEROUY et al., European Journal of Dermatology
no. 3, vol. 10, April-May 2000, pp. 173-180.
[0104] The metalloproteinase family thus consists of several
well-defined groups which are based on their similarity in terms of
structure and substrate specificity (see Woessner J. F., Faseb
Journal, vol. 5, 1991, 2145). Among these groups, mention may be
made of the collagenases which are intended to break down fibrillar
collagens (MMP-1 or interstitial collagenase, MMP-8 or neutrophil
collagenase, MMP-13 or collagenase 3 and MMP-18 or collagenase 4),
gelatinases which break down type IV collagen or any form of
denatured collagen (MMP-2 or gelatinase A (72 kDa) and MMP-9 or
gelatinase B (92 kDa)), stromelysins (MMP-3 or stromelysin 1,
MMP-10 or stromelysin 2 and MMP-11 or stromelysin 3), whose broad
spectrum of activity is aimed at proteins of the extracellular
matrix such as glycoproteins (fibronectin and laminin),
proteoglycans, etc., matrilysin (MMP-7), metalloelastase (MMP-12)
or else the membrane metalloproteinases (MMP-14, MMP-15, MMP-16 and
MMP-17).
[0105] The metalloproteinases (MMPs) are members of a family of
proteolytic enzymes (endoproteases) which possess a zinc atom which
is coordinated to 3 cysteine residues and a methionine in their
active site and which break down the macromolecular components of
the extracellular matrix and the basement membranes at neutral pH
(collagen, elastin, etc.). Being very widely distributed in the
living world, these enzymes are present, but weakly expressed, in
normal physiological situations such as the growth of the organs
and the renewal of the tissues. However, in humans, their
overexpression and their activation are linked to a large number of
processes which involve the destruction and remodeling of the
matrix. This leads, for example, to uncontrolled resorption of the
extracellular matrix.
[0106] A topical administration of this nature is useful to prevent
and/or combat signs of skin aging such as loss of firmness and/or
suppleness of the skin and/or atrophy of the skin and/or the
formation of wrinkles and fine lines; they are also suitable for
inducing and/or stimulating growth of head hair and/or body hair
and/or for retarding its loss.
[0107] In another embodiment of the invention, a double-stranded
RNA oligonucleotide is administered which is able to inhibit the
expression of a messenger RNA which encodes HMG-COA reductase.
[0108] In particular, the double-stranded RNA oligonucleotide is
such that the sequence of its 5' sense strand is SEQ ID no. 5:
5'-AGCCGCGAGGGUCGUCCAAdTdT and such that the sequence of its 5'
antisense strand is SEQ ID no. 6: 5'-UUGGACGACCCUCGCGGCUdTdT.
[0109] Such a topical administration is useful to prevent and/or
combat the excessive production of sebum or sweat in the region of
the armpits or the feet.
[0110] In another embodiment of the invention, a double-stranded
RNA oligonucleotide is administered which is able to inhibit the
expression of a messenger RNA which encodes type I and/or type II
5.alpha.-reductase, preferably type I 5.alpha.-reductase.
[0111] In particular, the double-stranded RNA oligonucleotide is
such that the sequence of its 5' sense strand is SEQ ID no. 7:
5'-CUGCAUCCUCCUGGCCAUGdTdT and such that the sequence of its 5'
antisense strand is SEQ ID no. 8: 5'-CAUGGCCAGGAGGAUGCAGdTdT.
[0112] Such a topical administration is useful to treat
androgen-dependent disorders, in particular to treat hyperseborrhea
and/or acne and/or to induce and/or stimulate growth of head hair
and/or body hair and/or retard loss of head hair.
[0113] In another embodiment of the invention, a double-stranded
RNA oligonucleotide is administered which is able to inhibit the
expression of a messenger RNA which encodes calmodulin-like skin
protein (CLSP).
[0114] In particular, the double-stranded RNA oligonucleotide is
such that the sequence of its 5' sense strand is SEQ ID no. 9:
5'-GGCUUUCUCCGCGGUUGACdTdT and such that the sequence of its 5'
antisense strand is SEQ ID no. 10: 5'-GUCAACCGCGGAGAAAGCCdTdT.
[0115] Such a topical administration is useful to combat the signs
of skin aging and/or combat the harmful effects of ultraviolet rays
and/or treat dry skin.
[0116] In another embodiment of the invention, a double-stranded
RNA oligonucleotide is administered which is able to inhibit the
expression of a messenger RNA which encodes NO synthase.
[0117] Such a topical administration is useful to inhibit cellular
proliferation and/or differentiation and/or inhibit the breakdown
and/or destruction of the cells of the skin and in this way combat
intrinsic and/or extrinsic aging and/or retard loss of the hair.
These compositions are also suitable for treating sensitive skin
and erythemas, in particular light-induced erythemas.
[0118] In another embodiment of the invention, a double-stranded
RNA oligonucleotide is administered which is able to inhibit the
expression of a messenger RNA which encodes a serine protease such
as urokinase. The compositions of this type are particularly
suitable for cosmetic treatment and for improving the appearance of
dry and/or irritated skin.
[0119] In other embodiments of the invention, a double-stranded RNA
oligonucleotide is administered which is able to inhibit the
expression of a messenger RNA encoding:
[0120] IL1, for its applications in the area of sensitive skin,
[0121] 5.alpha. reductase, for its applications in the area of
fatty skin,
[0122] a bacterial lipase, for its applications for combating
dandruff formation, or else
[0123] lox12 and/or cox2 and/or IL1 and/or TGF.beta.1, for
applications for combating hair loss.
[0124] For administration/application according to the invention,
the double-stranded RNA oligonucleotide can be present in a
composition at a concentration of from 5.10.sup.-7 to 5%,
preferably of from 0.0001 to 1% of double-stranded RNA
oligonucleotide(s) by weight based on the total weight of the
composition.
[0125] Such a composition can be present in all the galenic forms
which are normally employed for this type of application, in
particular in the form of an aqueous or oily solution, of an
oil-in-water or water-in-oil, or multiple, emulsion, of a
siliconated emulsion, of a microemulsion or nanoemulsion, of an
aqueous or oily gel or of an anhydrous liquid, pasty or solid
product.
[0126] The present invention also features compositions suited to
be applied topically and which comprise an encapsulated
double-stranded RNA oligonucleotide formulated into a
physiologically acceptable medium therefor.
[0127] In one preferred embodiment, the composition is such that
the double-stranded RNA oligonucleotide is encapsulated in the core
or the wall of a covering such as microspheres, nanospheres,
oleosomes, niosomes or nanocapsules.
[0128] The double-stranded RNA oligonucleotides, at an effective
molar concentration in an aqueous solution, can be complexed with a
polyethylene imine (PEI) having a molecular weight of from 200 to
100 000 g/m. The nature of the PEI, and its proportion of from
10.sup.-4 to 1%, depend on the initial type of aqueous solution
containing the double-stranded RNA oligonucleotides and on the
cosmetic formulation intended. The opalescence of the initial
solution can be used to indicate the formation of the complex.
[0129] Once the complex has been formed, it is encapsulated in
particles, such as niosomes, which have an aqueous core or it is
complexed on the surface of vesicles which have a lamellar surface
which contains a polymer or a cationic lipophilic surfactant such
as hydrophobic PEIs.
[0130] Thus, the double-stranded RNA oligonucleotide can be
either:
[0131] complexed by a polycation (for example a hydrophilic PEI or
chitosan);
[0132] By way of example, it is possible to select
poly(4-vinylpyridine)-d- erived polycations (Modulation of
interaction of polycations with negative unilamellar lipid
vesicles, by Yaroslavov et al. in Colloids and surfaces
B:Biointerfaces, 16(1999)-43); polymers and copolymers of lysine
(cited in Turbidometric analysis of polyelectrolyte complexes
formed between poly(L-lysine) and DNA, by Ward et al., in Colloids
and surfaces B:Biointerfaces, 16(1999)253-260); copolymers of
quaternized vinylimidazole and vinylpyrrolidine such as the
Luviquats from BASF (mw between 1000 and 1 000 000); chloride-based
copolymers of diallyl dimethylammonium such as the Merquats from
BASF (mw between 10 000 and 10 000 000); polyglucoseamines such as
chitosan and its derivatives; polyamines such as polyethyleneimine
which are or are not hydrophobic; guar derivatives such as the
Jaguars from Rhodia; cationic cellulose derivatives such as the
Celquats from National Starch or the Quarisofts from Amerchol;
copolymers of vinylpyrrolidone and dimethylaminopropylmeth-
acrylamide such as the Gafquats from ISP; cationic derivatives of
acrylic acid such as HYPAN QT 100 from Kingston; the Salcare SC92,
SC95 and 96 from Allied Colloide, and Plex4739L from Rohm Gmbh;
derivatives of diurethane and polyurethane such as Polyderme
PPI-SA, Foamox PPI-SA, Foamtaine PPI-SA15 and Foamquat PPI-SA from
Alzo and copolyamines such as polyquart H-81 from Henkel;
[0133] this complex can then be encapsulated in particles having an
aqueous core such as liposomes and, in particular, niosomes (see
EP-0,582,503 or U.S. Pat. No. 5,439,672);
[0134] complexed on the surface of vesicles which possess a
lamellar surface which contains a polymer or a cationic lipophilic
surfactant.
[0135] In this latter case, the cationic lipophilic surfactants can
be more particularly selected from the group consisting of
quaternary ammonium salts and fatty amines and their salts.
[0136] Examples of the quaternary ammonium salts are: 1
[0137] in which the radicals R.sub.1 to R.sub.4, which can be
identical or different, represent a linear or branched aliphatic
radical having from 1 to 30 carbon atoms or an aromatic radical
such as aryl or alkylaryl radicals. The aliphatic radicals can
comprise heteroatoms such as, in particular, oxygen, nitrogen,
sulfur and the halogens. The aliphatic radicals are selected, for
example, from the radicals alkyl, alkoxy,
polyoxy(C.sub.2-C.sub.6)alkylene, alkylamide,
(C.sub.12-C.sub.22)alkylami- do (C.sub.2-C.sub.6)alkyl,
(C.sub.12-C.sub.22)alkyl acetate and hydroxyalkyl having from about
1 to 30 carbon atoms; X is an anion which is selected from the
group of halides, phosphates, acetates, lactates,
(C.sub.2-C.sub.6)alkyl sulfates and alkyl- or alkylaryl-sulfonates.
Quaternary ammonium salts of the formula (II) which are preferred
are, on the one hand, tetraalkylammonium chlorides such as
dialkyldimethylammonium or alkyltrimethylammonium chlorides in
which the alkyl radical comprises from about 12 to 22 carbon atoms,
in particular the chlorides of behenyltrimethylammonium, of
distearyldimethylammonium, of cetyltrimethylammonium of
benzyldimethylstearylammonium, or else, on the other hand, the
chloride of stearamidopropyldimethyl(myristyl acetate)ammonium,
which is marketed by VAN DYK under the designation "CERAPHYL
70".
[0138] (2) The quaternary ammonium salts of imidazolinium, such as
those of the following formula: 2
[0139] in which R.sub.5 represents an alkenyl or alkyl radical
having from 8 to 30 carbon atoms, for example derived from tallow
fatty acids; R.sub.6 represents a hydrogen atom, an alkyl radical
having from 1 to 4 carbon atoms or an alkenyl or alkyl radical
having from 8 to 30 carbon atoms; R.sub.7 represents an alkyl
radical having from 1 to 4 carbon atoms; R.sub.8 represents a
hydrogen atom or an alkyl radical having from 1 to 4 carbon atoms;
X is an anion selected from the group consisting of halides,
phosphates, acetates, lactates, alkyl sulfates and alkyl- or
alkylarylsulfonates. Preferably, R.sub.5 and R.sub.6 denote a
mixture of alkenyl or alkyl radicals having from 12 to 21 carbon
atoms, for example derived from tallow fatty acids, R.sub.7 denotes
a methyl radical and R.sub.8 denotes hydrogen. Such a product is
marketed, for example, by REWO under the designation "REWOQUAT W
75".
[0140] (3) The quaternary diammonium salts of the formula: 3
[0141] in which R.sub.9 denotes an aliphatic radical having from
about 16 to 30 carbon atoms; R.sub.10, R.sub.11, R.sub.12, R.sub.13
and R.sub.14 are each hydrogen or an alkyl radical having from 1 to
4 carbon atoms; and X is an anion which is selected from the group
consisting of halides, acetates, phosphates, nitrates and methyl
sulfates. These quaternary diammonium salts comprise, in
particular, propanetallow diammonium dichloride.
[0142] The double-stranded RNA oligonucleotide can also be
complexed on the surface of cationic oily globules whatever their
size (see EP-1,010,413, EP-1,010,414, EP-1,010,415, EP-1,010,416,
EP-1,013,338, EP-1,016,453, EP-1,018,363, EP-1,020,219,
EP-1,025,898, EP-1,120,101, EP-1,120,102, EP-1,129,684,
EP-1,160,005 and EP-1,172,077).
[0143] The double-stranded RNA oligonucleotide can also be
complexed on the surface of nanocapsules or nanoparticles which are
provided with a lamellar coating (see EP-0,447,318 and
EP-0,557,489) and which contain a cationic surfactant at the
surface (see the previously cited references for the cationic
surfactants).
[0144] In particular, the vesicles which are preferred in
accordance with the present invention are:
[0145] the niosomes described in EP-0,582,503;
[0146] the lipid spherules whose method of synthesis is described
in U.S. Pat. No. 5,021,200;
[0147] the oil-in-water niosomes described in U.S. Pat. No.
5,489,426;
[0148] the nanoemulsions proposed in EP-0,879,589.
[0149] Preferred are the following lipids: PEI which is rendered
hydrophobic by grafting on C10 to C22 alkyl chains (from 5 to 50%),
which are unsaturated or saturated or branched or unbranched,
stearylamine or behenyltriammonium chloride.
[0150] More particularly, preferred are vesicles which have a
diameter which is less than or equal to 2 .mu.m and is preferably
between 50 nm and 1 .mu.m.
[0151] It is also possible to use physical methods of
intracutaneous administration such as a gas propulsion system or a
microneedle patch.
[0152] In FIG. 1, which depicts a microneedle patch, the minidarts
(cones) have a height which corresponds to the depth which the
dsRNA which is encapsulated in a vesicle should reach (superficial
epidermis, deep epidermis, superficial dermis, etc.). The size of
the patch can, for example, be matched to the area of a pigment
spot or a wrinkle.
[0153] In a more detailed manner, the composition which is applied
topically can be more or less fluid and have the appearance of a
white or colored cream, of an ointment, of a milk, of a lotion, of
a serum, of a paste, of a mousse or of a gel. It can, where
appropriate, be applied to the skin in the form of an aerosol. It
can also be present in solid form and, for example, be in the form
of a stick. It can be used as a care product and/or as a skin
makeup product.
[0154] Thus, the compositions according to the invention can, in
addition to the double-stranded RNA oligonucleotide, contain at
least one active compound selected from: .alpha.-hydroxyacids;
salicylic acid and its derivatives such as n-octanoyl-5-salicylic
acid; HEPES; procysteine; O-octanoyl-6-D-maltose; the disodium salt
of methylglycinediacetic acid; ceramides; steroids such as
diosgenin and derivatives of DHEA; kojic acid;
N-ethyloxycarbonyl-4-paraminophenol; ascorbic acid and its
derivatives; bilberry extracts; retinoids and, in particular,
retinol and its esters; polypeptides and their acylated
derivatives; phytohormones; extracts of the yeast Saccharomyces
cerevisiae; algal extracts; extracts of Vitreoscilla filiformis;
extracts of soybean, lupin, corn and/or pea; alverine and its
salts, in particular alverine citrate; resveratrol; carotenoids
and, in particular, lycopene; tocopherol and its esters; coenzyme
Q10 or ubiquinone; xanthines and, in particular, caffeine and the
natural extracts containing it; extracts of butcher's-broom and
horse-chestnut; and their mixtures, without this list being
limiting.
[0155] In addition, the compositions according to the invention can
contain at least one UVA and/or UVB filter. The sunscreen filters
can be selected from organic filters and inorganic filters and
their mixtures.
[0156] Examples of organic filters which are active in the UV-A
and/or the UV-B and which may in particular be mentioned are those
designated below by their CTFA name:
[0157] derivatives of paraminobenzoic acid: PABA, ethyl PABA,
ethyldihydroxypropyl PABA, ethylhexyldimethyl PABA, marketed, in
particular, by ISP under the name "ESCALOL 507", glyceryl PABA,
PEG-25 PABA, marketed by BASF under the name "UVINUL P25",
salicylic derivatives: homosalate, marketed by RONA/EM INDUSTRIES
under the name "EUSOLEX HMS", ethylhexyl salicylate, marketed by
HAARMANN and REIMER under the name "NEO HELIOPAN OS",
dipropyleneglycol salicylate, marketed by SCHER under the name
"DIPSAL", TEA salicylate, marketed by HAARMANN and REIMER under the
name "NEO HELIOPAN TS", derivatives of dibenzoylmethane:
butylmethoxydibenzoylmethane, marketed, in particular, by HOFFMANN
LA ROCHE under the trademark "PARSOL 1789",
isopropyldibenzoylmethane, cinnamic derivatives: ethylhexyl
methoxycinnamate, marketed, in particular, by HOFFMANN LA ROCHE
under the trademark "PARSOL MCX", isopropylmethoxycinnamate,
isoamylmethoxycinnamate, marketed by HAARMANN and REIMER under the
trademark "NEO HELIOPAN E 1000", cinoxate, DEA methoxycinnamate,
diisopropyl methylcinnamate, glyceryl ethylhexanoate
dimethoxycinnamate,
[0158] derivatives of .beta.,.beta.'-diphenylacrylate: octocrylene,
marketed, in particular, by BASF under the trademark "UVINUL N539",
etocrylene, marketed, in particular, by BASF under the trademark
"UVINUL N35",
[0159] derivatives of benzophenone: benzophenone-1, marketed by
BASF under the trademark "UVINUL 400", benzophenone-2, marketed by
BASF under the trademark "UVINUL D50", benzophenone-3 or
oxybenzone, marketed by BASF under the trademark "UVINUL M40",
benzophenone-4, marketed by BASF under the trademark "UVINUL MS40",
benzophenone-5, benzophenone-6, marketed by NORQUAY under the
trademark "HELISORB 11", benzophenone-8, marketed by AMERICAN
CYANAMID under the trademark "SPECTRA-SORB UV-24", benzophenone-9,
marketed by BASF under the trademark "UVINUL DS-49", and
benzophenone-12,
[0160] derivatives of benzylidene camphor: 3-benzylidene camphor,
4-methylbenzylidene camphor, marketed by MERCK under the name
"EUSOLEX 6300", benzylidene camphor sulfonic acid, camphor
benzalkonium methosulfate, terephthalylidene dicamphor sulfonic
acid and polyacrylamidomethyl benzylidene camphor, derivatives of
phenyl benzimidazole: phenylbenzimidazole sulfonic acid, marketed,
in particular, by MERCK under the trademark "EUSOLEX 232", and
benzimidazilate, marketed by HAARMANN and REIMER under the
trademark "NEOHELIOPAN AP",
[0161] derivatives of triazine: anisotriazine, marketed by CIBA
GEIGY under the trademark "TINOSORB S", ethylhexyl triazone,
marketed, in particular, by BASF under the trademark "UVINUL T150",
and diethylhexyl-butamidotriazone, marketed by SIGMA 3V under the
trademark "UVASORB HEB",
[0162] derivatives of phenyl benzotriazole: drometrizole
trisiloxane, marketed by RHODIA CHIMIE under the name
"SILATRIZOLE",
[0163] anthranilic derivatives: menthyl anthranilate, marketed by
HMRMANN and REIMER under the trademark "NEO HELIOPAN MA",
[0164] imidazoline derivatives:
ethylhexyldimethoxybenzylidenedioxoimidazo- line propionate,
[0165] derivatives of benzalmalonate: polyorganosiloxane having
benzalmalonate functions, marketed by HOFFMANN LA ROCHE under the
trademark "PARSOL SLX",
[0166] and their mixtures.
[0167] The organic UV filters which are more particularly preferred
are selected from among the following compounds:
[0168] ethylhexyl salicylate,
[0169] butylmethoxydibenzoylmethane,
[0170] ethylhexyl methoxycinnamate,
[0171] octocrylene,
[0172] phenylbenzimidazole sulfonic acid,
[0173] terephthalylidene dicamphor sulfonic,
[0174] benzophenone-3,
[0175] benzophenone-4,
[0176] benzophenone-5,
[0177] 4-methylbenzylidene camphor,
[0178] benzimidazilate,
[0179] anisotriazine,
[0180] ethylhexyl triazone,
[0181] diethylhexylbutamidotriazone,
[0182] methylene bis-benzotriazolyl tetramethylbutylphenol,
[0183] drometrizole trisiloxane,
[0184] and their mixtures.
[0185] The inorganic filters which can be used in the composition
according to the invention are, in particular, coated or uncoated
metallic oxide nanopigments (mean size of the primary particles: in
general, from 5 nm to 100 nm, preferably from 10 nm to 50 nm), such
as nanopigments of titanium oxide (amorphous or crystallized in
rutile and/or anatase form), of iron oxide, of zinc oxide, of
zirconium oxide or of cerium oxide. Coating agents are, in
addition, alumina and/or aluminum stearate. These coated or
uncoated metallic oxide nanopigments are described, in particular,
in EP-A,0-518,772 and EP-A,0-518,773.
[0186] In a known manner, the compositions of the invention can
also contain adjuvants which are customary in the cosmetic and
dermatological fields, such as hydrophilic or lipophilic
gelatinizing agents, preservatives, antioxidants, solvents,
surfactants, thickeners, perfumes, fillers, pigments, odor
absorbers and coloring substances. The quantities of these
adjuvants are those which are usually employed in the fields in
question and are, for example, from 0.01 to 20% of the total weight
of the composition. These adjuvants can be introduced, in
accordance with their nature, either into the fatty phase or into
the aqueous phase. These adjuvants, and their concentrations,
should be such that they do not impair the advantageous properties
of pantetheinesulfonic acid or its salts.
[0187] When the composition of the invention is an emulsion, the
proportion of the fatty phase can range from 5% to 80% by weight,
preferably from 5% to 50% of the total weight of the composition.
The oils, the emulsifiers and the coemulsifiers which are used in
the composition in emulsion form are selected from those which are
usually employed in the field in question. The emulsifier and the
coemulsifier are present, in the composition, in a proportion which
ranges from 0.3% to 30% by weight, preferably from 0.5% to 20% of
the total weight of the composition.
[0188] Examples of oils which can be used in the composition of the
invention and which may be mentioned are:
[0189] hydrocarbonaceous oils of animal origin, such as
perhydrosqualene;
[0190] hydrocarbonaceous oils of vegetable origin, such as liquid
fatty acid triglycerides which comprise from 4 to 10 carbon atoms
and the liquid fraction of karite butter;
[0191] synthetic esters and ethers of, in particular, fatty acids,
such as the oils of the formulae R.sup.1COOR.sup.2 and
R.sup.1OR.sup.2 in which R.sup.1 represents the residue of a fatty
acid comprising from 8 to 29 carbon atoms and R.sup.2 represents a
branched or unbranched hydrocarbon chain which contains from 3 to
30 carbon atoms, such as Purcellin's oil, isononyl isononanoate,
isopropyl myristate, ethyl-2-hexyl palmitate, octyl-2-dodecyl
stearate, octyl-2-dodecyl erucate, and isostearyl isostearate;
hydroxylated esters such as isostearyl lactate,
octylhydroxystearate, octyldodecyl hydroxystearate,
diisostearylmalate, triisocetyl citrate, and heptanoates,
octanoates and decanoates of fatty alcohols; polyol esters, such as
propylene glycol dioctanoate, neopentylglycol diheptanoate and
diethyleneglycol diisononanoate; and pentaerythritol esters, such
as pentaerythrityl tetraisostearate;
[0192] linear or branched hydrocarbons of mineral or synthetic
origin, such as volatile or nonvolatile paraffin oils and their
derivatives, petrolatum, polydecenes, and hydrogenated
polyisobutene such as parleam oil;
[0193] fatty alcohols having from 8 to 26 carbon atoms, such as
cetyl alcohol and stearyl alcohol and their mixture (cetylstearyl
alcohol), octyldodecanol, 2-butyloctanol, 2-hexyldecanol,
2-undecylpentadecanol, oleic alcohol or linoleic alcohol;
[0194] partially hydrocarbonaceous and/or siliconaceous fluorinated
oils, such as those described in JP-A-2-295912;
[0195] silicone oils such as volatile or nonvolatile
polymethylsiloxanes (PDMS) which have a linear or cyclic
siliconaceous chain and which are liquid or pasty at ambient
temperature, in particular cyclopolydimethylsiloxanes
(cyclomethicones) such as cyclohexasiloxane; polydimethylsiloxanes
which comprise alkyl, alkoxy or phenyl groups which are pendent or
at the end of the siliconaceous chain, with the groups having from
2 to 24 carbon atoms; phenylated silicones such as
phenyltrimethicones, phenyldimethicones,
phenyltrimethylsiloxydiphenylsil- oxanes, diphenyldimethicones,
diphenylmethyldiphenyltrisiloxanes,
2-phenylethyltrimethylsiloxysilicates and
polymethylphenylsiloxanes;
[0196] their mixtures.
[0197] Examples of emulsifiers and coemulsifiers which can be used
in the invention and which may be mentioned are ONV emulsifiers
such as esters of fatty acid and polyethylene glycol, in particular
PEG-100 stearate, and esters of fatty acid and glycerol, such as
glyceryl stearate, as well as W/O emulsifiers such as the
oxyethylenated poly(methylcetyl)(dimethyl)- methylsiloxane which is
available from Degussa Goldschmidt under the trademark ABIL WE09,
or the mixture of ethylene glycol acetyl stearate and glyceryl
tristearate which is marketed by Guardian under the trademark
UNITWIX.
[0198] Hydrophilic gelatinizing agents which may be mentioned, in
particular, are carboxyvinylic polymers (carbomer), acrylic
polymers such as acrylate/alkyl acrylate copolymers,
polyacrylamides, polysaccharides, natural gums and clays, while
lipophilic gelatinizing agents which may be mentioned are modified
clays such as bentonites, metallic salts of fatty acids,
hydrophobic silica and polyethylenes.
[0199] Examples of fillers which may be employed in the composition
of the invention and which may be mentioned are, in addition to
pigments, silica powder; talc; starch which is crosslinked with
octenylsuccinic anhydride and which is marketed by National Starch
under the designation DRY FLO PLUS (28-1160); polyamide particles,
in particular those sold by Atochem under the designation ORGASOL;
polyethylene powders; microspheres based on acrylic copolymers,
such as those made of ethylene glycol dimethacrylate/lauryl
methacrylate copolymer and sold by Dow Corning under the
designation POLYTRAP; expanded powders such as hollow microspheres
and, in particular, the microspheres marketed by Kemanord Plast
under the designation EXPANCEL, or by Matsumoto under the
designation MICROPEARL F 80 ED; silicone resin microbeads such as
those marketed by Toshiba Silicone under the designation TOSPEARL;
and their mixtures. These fillers can be present in quantities
ranging from 0 to 20% by weight, and preferably of from 1 to 10% by
weight, based on the total weight of the composition or of the
preparation according to the invention.
[0200] One skilled in this art will take care to use ingredients
and active compounds which do not impair the activity of the
double-stranded RNA oligonucleotides which are present in the
compositions according to the invention.
[0201] The present invention also features a cosmetic treatment
method (regime or regimen) which comprises topically applying, to
an area to be treated, a composition which comprises at least one
double-stranded RNA oligonucleotide, as previously defined, in a
physiologically acceptable medium. Advantageously, the composition
is applied to the skin and/or the scalp and/or the hair and nails
either once only or repeatedly over time.
[0202] The method can be a method for depigmenting and/or bleaching
the skin or the nails and hair, with the method being characterized
in that a composition according to the invention, in particular a
composition which comprises a double-stranded RNA oligonucleotide
which is able to inhibit the expression of a messenger RNA which
encodes tyrosinase and/or a double-stranded RNA oligonucleotide
which is able to inhibit the expression of a messenger RNA which
encodes TRP-1, is applied to the area to be treated.
[0203] This invention also features a cosmetic treatment method for
preventing and/or combating cutaneous signs of aging, with this
method being characterized in that a composition comprising a
double-stranded RNA oligonucleotide which is able to inhibit the
expression of a messenger RNA which encodes neutrophil elastase
and/or a double-stranded RNA oligonucleotide which is able to
inhibit the expression of a messenger RNA which encodes
hyaluronidase and/or a double-stranded RNA oligonucleotide which is
able to inhibit the expression of a messenger RNA which encodes a
metalloproteinase and/or a double-stranded RNA oligonucleotide
which is able to inhibit the expression of a messenger RNA which
encodes HMG-CoA reductase and/or a double-stranded RNA
oligonucleotide which is able to inhibit the expression of a
messenger RNA which encodes calmodulin-like skin protein (CLSP)
and/or a double-stranded RNA oligonucleotide which is able to
inhibit the expression of a messenger RNA which encodes NO synthase
is applied to the area to be treated.
[0204] The present invention also features a cosmetic treatment
method for inducing and/or stimulating growth of head hair and/or
body hair and/or for retarding loss of head hair, with this method
being characterized in that a composition comprising a
double-stranded RNA oligonucleotide which is able to inhibit the
expression of a messenger RNA which encodes a metalloproteinase
and/or a double-stranded RNA oligonucleotide which is able to
inhibit the expression of a messenger RNA which encodes type I or
type II 5.alpha.-reductase and/or a double-stranded RNA
oligonucleotide which is able to inhibit the expression of a
messenger RNA which encodes NO synthase is applied to the area to
be treated.
[0205] In order to further illustrate the present invention and the
advantages thereof, the following specific examples are given, it
being understood that same are intended only as illustrative and in
nowise limitative. In said examples to follow, all parts and
percentages are given by weight, unless otherwise indicated.
[0206] In these examples, reference will be made to the following
figures of drawing:
[0207] FIG. 1: depicts a microneedle patch which can be used for
administering double-stranded RNA oligonucleotides.
[0208] FIG. 2: specific extinction, by RNA interference, of the
expression of tyrosinase in human melanocytes (MeWo).
EXAMPLE 1
Using RNA Interference to Inhibit the Expression of Tyrosinase:
[0209] Materials and Methods:
[0210] The experimental conditions are those described by Tuschl et
al. (Journal of Cell Science, 2001, 114, 45574565).
[0211] 24 h before transfection, the melanocytes (MeWo cells) are
sown, at the rate of 40,000 cells/well, in 500 .mu.l of DMEM
medium+10% serum in a 24-well plate. 0.84 .mu.g or 2.52 .mu.g of
duplex siRNA (see description below), that is 60 pmol of tyrosinase
siRNA in 3 .mu.l of annealing buffer (Dharmacon) or, respectively,
180 pmol of tyrosinase siRNA in 9 .mu.l of annealing buffer, are
used per well. 3 .mu.l or 9 .mu.l of 20 .mu.M tyrosinase duplex
siRNA are diluted in 50 .mu.l of Opti-MEM (Gibco). In another tube,
3 .mu.l of oligofectamine (Invitrogen) are diluted in 12 .mu.l of
Opti-MEM and incubated at room temperature for 10 minutes. The 2
solutions are mixed and incubated at room temperature for 20
minutes before being added to the cells. The cells are then
incubated at 37.degree. C. for 96 h, with a change of medium after
24 h, and then lysed in a buffer containing 10 mM Tris-HCl, pH 7.2,
2% SDS, 1% Triton X-100,10% glycerol+1% protease inhibitor; 34
.mu.g of protein are then added, per well, to a polyacrylamide gel
(NuPAGE Novex bis-Tris gels, Invitrogen), separated by
electrophoresis and transferred by Western blotting in accordance
with the supplier's instructions. The presence of tyrosinase on the
membrane is analyzed by hybridizing with an antityrosinase antibody
diluted 1/500 (NCL-TYR, Novocastra), and the quantity of protein
which has been added is checked by hybridizing with an antivimentin
antibody which has been diluted 1/40 000 (VIM3B4 clone, Cymbus
Biotechnologies). The membrane is rinsed in PBS-Tween 20 and
hybridized with a peroxidase-coupled antimouse secondary antibody
(Goat antimouse-HRP, Dako) diluted 1/2000. Detection is effected
using an "ECL Plus western blotting detection system" kit and
following the supplier's (Amersham) instructions.
[0212] The following siRNA duplexes are selected from the sequence
encoding human tyrosinase cDNA (accession number M27160). The
adjacent number corresponds to the position of the 1.sup.st
nucleotide in the nucleic acid sequence. The duplexes are
synthesized by Dharmacon or from DNA oligonucleotides provided by
Proligo and transcribed into RNA using a "siRNA construction kit"
(Ambion) and following the supplier's instructions. The 7 duplexes
comprising an RNA sense strand and an RNA antisense strand consist
of the following sequences, where AUG are ribonucleic acid bases
and dT is a deoxyribonucleic acid base:
1 siRNA-TYR266 duplex: siRNA 5' sense strand (SEQ ID N.sup.o1):
UGCACCACUUGGGCCUCAAdTdT siRNA 5' antisense strand (SEQ ID
N.sup.o2): UUGAGGCCCAAGUGGUGCAdTdT siRNA-TYR359 duplex: siRNA 5'
sense strand (SEQ ID N.sup.o11): AAGUGUUUGAUGCUGGAGGdTdT siRNA 5'
antisense strand (SEQ ID N.sup.o12): CCUCCAGCAUCAAACACUUdTdT
siRNA-TYR69O duplex: siRNA 5' sense strand (SEQ ID N.sup.o13)::
GCACCAGCUUUUCUGCCUUdTdT siRNA 5' antisense strand (SEQ ID
N.sup.o14): AAGGCAGAAAAGCUGGUGCdTdT siRNA-TYR832 duplex: siRNA 5'
sense strand (SEQ ID N.sup.o15): ACUGCACAGAGAGACGACUdTdT siRNA 5'
antisense strand (SEQ ID N.sup.o16): AGUCGUCUCUCUGUGCAGUdTdT
siRNA-TYR1110 duplex: siRNA 5' sense strand (SEQ ID N.sup.o17):
GCACCAGCUUUUCUGCCUUdTdT siRNA 5' antisense strand (SEQ ID
N.sup.o18): AAGGCAGAAAAGCUGGUGCdTdT siRNA-TYR1578 duplex: siRNA 5'
sense strand (SEQ ID N.sup.o19): AGCAGCAUGCACAAUGCCUdTdT siRNA 5'
antisense strand (SEQ ID N.sup.o20): AGGCAUUGUGCAUGCUdTdT
siRNA-TYR2120 duplex: siRNA 5' sense strand (SEQ ID N.sup.o21):
AGCCUGACCUCACUCUAACdTdT siRNA 5' antisense strand (SEQ ID
N.sup.o22): GUUAGAGUGAGGUCAGGCUdTdT
[0213] The results are depicted in FIG. 2.
[0214] The lanes correspond to the following products:
[0215] L: molecular weight ladder
[0216] C: MeWo control
[0217] 1: MeWo+TYR266 siRNA, 0.84 .mu.g
[0218] 2: MeWo+TYR266 siRNA, 2.52 .mu.g
[0219] 3: MeWo+TYR359 siRNA, 0.84 .mu.g
[0220] 4: MeWo+TYR359 siRNA, 2.52 .mu.g
[0221] 5: MeWo+TYR690 siRNA, 0.84 .mu.g
[0222] 6: MeWo+TYR690 siRNA, 2.52 .mu.g
[0223] 7: MeWo+TYR832 siRNA, 0.84 .mu.g
[0224] 8: MeWo+TYR832 siRNA, 2.52 .mu.g
[0225] 9: MeWo+TYR1110 siRNA, 0.84 .mu.g
[0226] 10: MeWo+TYR1110 siRNA, 2.52 .mu.g
[0227] 11: MeWo+TYR1578 siRNA, 0.84 .mu.g
[0228] 12: MeWo+TYR1578 siRNA, 2.52 .mu.g
[0229] 13: MeWo+TYR2120 siRNA, 0.84 .mu.g
[0230] 14: MeWo+TYR2120 siRNA, 2.52 .mu.g
[0231] The extinction of tyrosinase expression is confirmed by
Western blotting. It appears that, of the 7 duplexes selected, 1
duplex is ineffective (359 siRNA), while 1 duplex is weakly
effective (1578 siRNA) and the other 5 duplexes are functional,
with what is apparently very powerful efficacy in the case of
duplexes 1110 and 2120, as is demonstrated by the failure to detect
tyrosinase. Specific inhibitors of tyrosinase are thus used to
inhibit, by means of "RNA interference", the expression of proteins
by epidermal cells.
Example 2
Compositions:
[0232] Composition 1: The double-stranded RNA oligonucleotide is
complexed on the surface of cationic vesicles:
[0233] Preparing the vesicles:
2 PEG 400 isostearate 8% Polyethyleneimine (PEI of 200 to 100 000
MW) 2% amidified with isostearic acid Distilled water qs for
100%
[0234] Double-stranded RNA oligonucleotides of SEQ ID No. 1 and SEQ
ID No. 2, with a dsRNA/PEI ratio of between 0.1 and 1000.
[0235] Steps Involved in Preparing Vesicles:
[0236] solubilizing the lipids in a methanol/chloroform
mixture;
[0237] evaporating the solvent in a rotary evaporator under reduced
pressure in order to obtain a lipid film;
[0238] hydrating the film while ultrasonicating;
[0239] checking the diameter of the vesicles (less than 1 .mu.m;
preferably 200 nm);
[0240] adding the solution of double-stranded RNA
oligonucleotides.
[0241] Cosmetic Composition:
[0242] The vesicles are then incorporated into a cosmetic
formulation which is suitable for topical application.
[0243] Daily application, preferably in the evening, of this
preparation attenuates pigmentary spots; regular and long-term
application leads to the disappearance of these pigmentary
spots.
[0244] Composition 2: The dsRNA is complexed in the interior of
cationic vesicles:
[0245] A) Encapsulating a Polycation/Double-Stranded RNA
Oligonucleotides Complex:
3 POE (8) stearate 5.5% Cholesterol 4% Polyethyleneimine (200 to
100 000 MW), 0.5% amidified with isostearic acid
[0246] Aqueous solution A: distilled water, 30%, PEI (200 to 100
000 MW), 1%, and double-stranded RNA oligonucleotide, QS to make
up.
[0247] Aqueous solution B: distilled water, qs for 100%
[0248] B) Steps for preparing the vesicles:
[0249] See the preparation of composition 1. In this case, the
hydration step is effected with ultrasonication, at the temperature
which is suitable for the lipid mixture and with solution A, and a
step of diluting with aqueous solution B is then added.
[0250] Cosmetic Composition:
[0251] The vesicles are then incorporated into a cosmetic
formulation which is suitable for a topical application.
[0252] In this manner, compositions 1 and 2 can then be introduced,
in a manner which is customary to one skilled in the art, into the
following formulation. The ingredients are identified in accordance
with the CTFA nomenclature.
4 Glycerol 5% Preservatives 1% Disodium EDTA 0.2% Carbomer 0.5%
Triethanolamine 0.25% Ammonium polyacryloyldimethyltaurate* 0.3%
Sodium hyaluronate 0.1% Vegetable oils 3% Acrylamide/sodium
acryloyldimethyltaurate 2% and isododecane and polysorbate-80**
Cyclopentasiloxane and dimethiconol 3% Vesicle suspension in
accordance with 1 to 25% example 1 or 2 Water qs for 100%
*Hostacerin AMPS supplied by CLARIANT **Simulgel 600 supplied by
SEPPIC
[0253] Each patent, patent application, publication and literature
article/report cited or indicated herein is hereby expressly
incorporated by reference.
[0254] While the invention has been described in terms of various
specific and preferred embodiments, the skilled artisan will
appreciate that various modifications, substitutions, omissions,
and changes may be made without departing from the spirit thereof.
Accordingly, it is intended that the scope of the present invention
be limited solely by the scope of the following claims, including
equivalents thereof.
Sequence CWU 1
1
22 1 21 DNA Artificial Sequence Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide 1 ugcaccacuu gggccucaat t 21 2
21 DNA Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 2 uugaggccca aguggugcat t 21 3 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 3 cggcuacgac cccguaaact t 21 4 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 4 guuuacgggg ucguagccgt t 21 5 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 5 agccgcgagg gucguccaat t 21 6 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 6 uuggacgacc cucgcggcut t 21 7 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 7 cugcauccuc cuggccaugt t 21 8 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 8 cauggccagg aggaugcagt t 21 9 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 9 ggcuuucucc gcgguugact t 21 10 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 10 gucaaccgcg gagaaagcct t 21 11 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 11 aaguguuuga ugcuggaggt t 21 12 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 12 ccuccagcau caaacacuut t 21 13 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 13 gcaccagcuu uucugccuut t 21 14 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 14 aaggcagaaa agcuggugct t 21 15 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 15 acugcacaga gagacgacut t 21 16 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 16 agucgucucu cugugcagut t 21 17 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 17 gcaccagcuu uucugccuut t 21 18 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 18 aaggcagaaa agcuggugct t 21 19 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 19 agcagcaugc acaaugccut t 21 20 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 20 aggcauugug caugcugcut t 21 21 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 21 agccugaccu cacucuaact t 21 22 21 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule
Synthetic oligonucleotide 22 guuagaguga ggucaggcut t 21
* * * * *