WO2006036082A1

WO2006036082A1 - Cosmetic agent and a method for the production thereof

Info

Publication number: WO2006036082A1
Application number: PCT/RU2004/000347
Authority: WO
Inventors: Artur Vladimirovich Rybakin
Original assignee: Obschestvo S Ogranichennoy Otvetstvennostyu 'sankt-Peterburgsky Institut Krasoty'
Priority date: 2004-09-06
Filing date: 2004-09-06
Publication date: 2006-04-06

Abstract

The invention relates to a cosmetic agent for improving a skin appearance and containing an active ingredient and a carrier, wherein said active ingredient comprises a complex containing a DNA telomeric sequence and a peptide which is bound therewith, provided with a nuclear localisation signal and penetrates through a cell plasmatic membrane.

Description

COSMETIC PRODUCT AND METHOD FOR ITS PRODUCTION

Technical field

The present invention relates to the field of cosmetology and relates to skin care products.

State of the art

Numerous cosmetics are known to enhance the appearance of the skin. In particular, cosmetics are known, the use of which allows you to accumulate and retain water in the skin, thereby eliminating or preventing dryness and loss of skin elasticity for some time.

Known cosmetics containing antioxidants that bind free radicals formed in the skin or prevent the formation of new ones, thereby preventing the occurrence of reactions with their participation that adversely affect the normal functioning of the cell. For example, drugs are known that contain ascorbic acid or vitamin E as antioxidants (Holsk David E. E., Jon D. Na, Fasial skip reptiviop, Cyr Orip and Orthal 14: 249). Known cosmetics containing substances

(e.g. retinoids (Nolsk David EE, Joh D. D. Λ / q, Facial skip diet, Hypalpol 14: 248), which stimulate fibroblast activity and the synthesis of new collagen fibers in the dermis, as well as mitotic activity and cell renewal, leading to a decrease in the thickness of the stratum corneum, an increase in the thickness of the granular layer of the skin and an improvement in the structure of the skin.

A disadvantage of the known cosmetics is that they do not affect the aging process of the skin cells of the body, giving only a relatively small effect on improving its functions and appearance. Disclosure of invention

An object of the present invention is to provide a cosmetic product capable of preventing aging of skin cells, thereby improving skin function and appearance more effectively.

This problem is solved by the fact that the proposed cosmetic product containing the active ingredient and the carrier, in which, according to the invention, the active ingredient contains a complex that includes a telomeric DNA sequence and its associated peptide having a nuclear localization signal (NLS), and which is able to penetrate across the plasma membrane of the cell.

When using the proposed cosmetic product, the telomeric DNA sequence contained in it penetrates the nucleus of the skin cell, leading to an increase in the number of divisions that the cell is able to carry out, i.e. slowing down the aging process of the cell, as a result of which the skin acquires a large number of young cells. This ensures the acceleration of re-epithelialization of the skin, an increase in the density of its cells and counteraction to thinning of the skin in the epidermis, as well as an increase in the production of elastin fibers by fibroblasts in the dermis. The result is a more effective improvement in the appearance of the skin, as well as its more efficient functioning.

To form a complex capable of penetrating the plasma membrane of a cell, said peptide contained in a cosmetic product of the invention may include a protein transduction domain (PTD). Such peptides may contain the amino acid sequences GRKKRRQRRRPPQKKKKKKV, RQIKIWFQNRRKKKKKKKKKKK OR

GALFLGFLGААGSТМGАWSQРКSКRКV.

The indicated ability to penetrate the plasma membrane of a cell can also be achieved by using a complex including a liposome into which the indicated telomeric DNA sequence and the indicated peptide are introduced. This problem is also solved by the fact that the proposed method of obtaining a cosmetic product, including the synthesis of the telomeric sequence of human DNA and the connection of the obtained telomeric sequence with a peptide having a nuclear localization signal, with the formation of a complex that can penetrate through the plasma membrane of the cell.

To form a complex, a peptide containing a protein transduction domain can be used. According to another embodiment of the invention, when the complex is formed, the telomeric DNA sequence and the peptide associated with it are introduced into the liposome.

According to another embodiment of the invention, upon receipt of a cosmetic product, the telomeric sequence is synthesized based on the total genomic DNA used as a matrix, which is obtained from a blood sample or human tissue to which the cosmetic product is intended to be applied.

The proposed cosmetic product contains the active ingredient and a carrier. The active ingredient is a complex that includes a telomeric DNA sequence and is able to penetrate the plasma membrane of the cell. The complex also contains a peptide linked to a telomeric sequence b having a nuclear localization signal.

Telomeric regions, or telomeres, of DNA chromosomes are special terminal regions of linear chromosomal DNA, to which certain proteins are also associated. It is believed that telomeres, in particular, protect chromosomes from degradation and fusion with other chromosomes. Telomeric DNA consists of repeatedly repeated short tandem nucleotide sequences, where one DNA strand is enriched in guanine (G), and its complementary strand is enriched in cytidine (C). In humans, like most eukaryotes, G-rich the telomeric DNA chain is constructed from TTAGGG blocks, and the C-rich chain contains CCCTAA blocks.

The proposed cosmetic product preferably includes telomeric sequences of human DNA containing from 100 to 300 p. However, sequences of a different length can also be used according to the present invention.

A nuclear localization signal refers to an amino acid sequence, also called NLS (from the English expression “nuclear losalysis sequence”), recognized by certain receptors on the cell’s nuclear membranes that carry the peptide containing it from the cytoplasm into the nucleus. The NLS sequences are described, for example, in Japs D. A. et al., Nisleart tarapetipa resorpShop: And kev soptrol poipt and postpaprrt? 2000, BioEssavs 22: 532-544. NLS sequences include, in particular, the NLS sequence of the T-antigen (T-ant) of the SV-40 virus, consisting of PKKKRKV amino acids, which are recognized and associated with a number of receptors of the nuclear membranes of human cells involved in the transport of peptides into the nucleus, and some similar with it amino acid sequences. (Japs DA et al., Siqpalsi teparata apde thеir resiiliatiop: Arrlisatiip dría deléveru. Med Res Rév 1998; 18: 189-223; Li S, Ku CY. Fépert. Of course, I suturedlasstis roteip tаt sіrtifiсallu bіpds thаt wrote a losalizаtiop siаpal totifs. J Віl Сhet 1998; 273: 6183-6189).

In addition to the NLS sequence, the peptides that make up this complex may contain other amino acid sequences.

Obtaining a complex capable of penetrating through the plasma membrane of a cell can be achieved by including in it any components giving this property. So, for example, protein sequences called protein transduction domains, also known as PTDs (from the expression “protein truncation domain”), or “penetrating into the cell of the peptides. " The protein transduction domain gives peptides having such domains the ability to penetrate the plasma membrane of cells. A number of PTD amino acid sequences are known (see, for example, Kaboyridis PS, Biodisal arrlisatiop of prototype tripsol, TRENDS ip Biolav, vol. 21, No.11, Novetber 2003, 498-503). Such PTD sequences that can be used in accordance with the present invention include, in particular, a peptide corresponding to amino acids 48-60 of the TAT protein of human immunodeficiency virus (HIV), having the amino acid sequence GRKKRRQRRRPPQ (GreepM apd Lowepstep PM (1998) Automated dothaips of chetisallu suptétizéd hitap ittipodefíciepsv virís tat trips-activator roroteip. CeII 55, 1179-1188). Another such PTD sequence is a peptide corresponding to amino acids 43-58 of the protein of the Antennapedia family of homeoboxic proteins (Apt), found in Dsorchil, and having the amino acid sequence RQIK1WFQNRRMKWKK

(Kaboiridis, see above).

Such PTD amino acid sequences can be included in a peptide linked to a telomeric sequence and carrying NLS. Thus, peptides that can be used in the complex contained in the cosmetic product of the invention are, for example,

GRKKRRQRRRPPQKKKKRKV, which is a fusion protein containing PTD, which is a 13-amino acid sequence of TAT (48-60), and NLS, which is a sequence of 7 amino acids T-ag SV-40. Another peptide that can be used to create this complex is RQIKIWFQNRRMKWKKKKKKRKV, which is a fusion protein containing 16-amino acid PTD Aptr (43-58) and 7-amino acid NLS T-ag SV-40. Another such peptide is MPG, which is a 27-amino acid peptide GALFLGFLGAAGGTMTGGWSQРКSКРКV, CONSIDERING OF HYDROPHOBIC The PTD domain, which is the sequence of HIV-1 gp41, (amino acids 1-16), and the hydrophilic domain (amino acids 20-27), which is T-ag SV-40 (see also Morris MC, Chalip L Merv J Heitz. F apd Divita G. And then I finished the song strateav fore delivep / sipa and sipale reptide sargier, 1999. Nislés As Res. 27. 3510-3517).

The association of telomeric sequences with a peptide in a complex contained in a cosmetic product according to the invention can be of a different nature. Peptides containing NLS and PTD carry a positive charge and can directly bind to negatively charged DNA. Chemical bonds between telomeric DNA and peptides can be formed with the participation of chemical groups both already present in these molecules and introduced into them by known modifications, for example, using the biotin-streptavidin system. Other substances besides PTD can be used to allow the complex of the invention to enter the cell. Thus, in another embodiment of the present invention, the complex of telomeric DNA and peptide having NLS contained in the cosmetic product of the invention further includes a liposome into which said telomeric DNA and said peptide are introduced.

Liposomes are vesicles having membranes that consist of lipid bilayers, mainly phospholipids, and which contain an internal aqueous phase. Liposomes are widely used in medicine and cosmetology to deliver the substances enclosed in them into cells.

A cosmetic product according to the invention may contain both single and multi-layer liposomes. Liposomes useful in such a cosmetic product may contain traditional ingredients. So, lipids are mainly phospholipids and their derivatives. Commonly used lipids include, in particular, phosphatidylcholine, cholesterol, phosphatidylethanolamine, dioleylphosphatidylglycerol, dioleylphosphatidylethanolamine and others. The aqueous phase in the liposomes contained in the cosmetic product according to the invention, in addition to the telomeric DNA bound peptides, can also contain auxiliary ingredients traditional for liposomes, for example buffers, antioxidants (for example sodium ascorbate) and chelating agents (for example ethylenediaminetetraacetic acid).

Carriers that can be used as part of the proposed cosmetic products are buffers, bases for ointments and creams, and other traditional carriers for cosmetic preparations. Carriers may contain buffering agents, gelling agents, preservatives, and other excipients used in the art.

The proposed method of obtaining a cosmetic product includes the synthesis of a telomeric sequence of human DNA and the connection of the obtained telomeric DNA sequence with a peptide having a nuclear localization signal, with the formation of a complex that can penetrate through the plasma membrane of the cell.

The telomeric sequence of human DNA can be obtained and amplified by polymerase chain reaction (PCR) using total genomic DNA isolated from human body tissues as a seed. For example, it is convenient to obtain DNA from human blood cells. Total genomic DNA is isolated from human tissue, in particular from blood leukocytes, by known methods, for example, by phenol-chloroform extraction. Selection of conditions for PCR is a routine procedure and is carried out by known methods. To generate telomeric DNA, the PCR product can be amplified again. From a pool of telomeric sequences obtained in PCR of different lengths, sequences of the desired length are isolated, for example, by gel electrophoresis followed by extraction DNA from the gel, while the selection of conditions for such isolation is also a routine for specialists.

Cloning in plasmids can also be used to generate additional numbers of telomeric DNA sequences and their storage. Suitable plasmids include, for example, pCR 2.1. Cloning is conveniently performed using the TA-cloning kit (IPvitrodep).

Peptides containing NLS, as well as peptides containing both NLS and PTD, for example, as described above, can be obtained, for example, by chemical synthesis.

The formation of a complex of telomeric DNA sequences with a peptide having NLS is carried out, for example, by introducing both components into a carrier, which is a buffer solution having a pH close to 7.0. The preferred molar ratio of the peptide to telomeric sequences is 100: 1 and higher. Electrophoresis in a 1% agarose gel followed by staining with ethidium bromide and UV light photography allows detection of a delay in the migration of DNA fragments in the gel, which indicates the formation of the complex. In order to obtain a cosmetic product in accordance with the invention, when the complex is formed, the telomeric DNA sequence and peptide can be introduced into the liposome. The formation of liposomes containing DNA associated with the peptide is carried out according to standard methods. Liposomes containing these active substances are obtained, for example, by dispersing lipids and DNA / peptide complexes in an aqueous medium, followed by mechanical stirring, sonication and / or extrusion through filters with a defined pore size. Liposomes can also be obtained by solubilization of lipids and active substances in organic solvents or detergents, mixing with an aqueous medium and subsequent removal of the solvent, as well as other known methods. Example

Total DNA was isolated from human blood leukocytes (0.5 ml whole blood) using the standard phenol-chloroform extraction method. The DNA yield after extraction was 40-50 μg. Amplification of DNA ^repeats' telomere was performed by polymerase chain reaction (PCR) which is carried out in a 30 .mu.l reaction mixture containing 67 mM Tris-HCL (pH 8.8), 16.6 mM (NH4) 2SO4, 0.1% Tritop X-100, 2.0mM MgCI_2, 0.5% dimethyl sulfoxide (DMSO), 4% formamide, 3 μl 25 mM dNTP, 3 units of Taq DNA polymerase, 2 μg of isolated genomic DNA, 30 pmol of direct (Forward) primer (TTAGGG) ₅ , 20 pmol of reverse (Reverse) , (AATCCC) ₅ . Both primers were obtained according to the technique proposed by Lorite et al., J. of Finalit 2002: 93 (4). PCR was performed on a thermocycler using a hot stagt. After initial denaturation at 94 ° C for 3 min with the Forward primer, Taq polymerase was introduced and incubated for an additional 7 min at the same temperature. Then, 10 amplification cycles were performed in the following mode: melting at 94 ° C for 1 min, annealing at 70 ° C for 1 min, synthesis of 72 ° C for 1 min. After completion of 10 cycles, a pause of 9O ⁰ C was established for 10 minutes, the Reverse primer was introduced. Then, 35 cycles of amplification were carried out in the same temperature-time regime as for the previous 10 cycles. After completing 35 amplification cycles, the final synthesis was performed at 72 ° C for 7 min.

1 μl of the obtained PCR product was taken, diluted with water in a ratio of 1/100, and 1 μl of the resulting solution was added to the reaction mixture for new PCR, which was carried out under the conditions and conditions described above.

20 μg of the PCR product obtained after reamplification of the reaction mixture was subjected to electrophoresis in 1% agarose gel containing TAE (Tris-acetate buffer, 40 mM Tris-base, 20 mM acetic acid, 1 mM EDTA) at a gradient of 8 V / cm. After electrophoresis, the gel was stained with ethidium bromide and photographed in UV light. As markers of molecular weight, kits of the company Stratagep (USA) were used.

Strips of the agarose gel corresponding to fragments of telomeric DNA 100-300 bp was excised from the gel and isolation of DNA from them was carried out using a set of ^'Qiagep (Qiagep) according to the manufacturer's instructions. Received 20 μl of a solution containing

100 ng of telomeric DNA sequences.

Then formed a complex of telomeric DNA with a peptide

GRKKRRQRRRRPQRKKKRKV (described above), produced by the Institute for Extra Pure Drugs (St. Petersburg), by adding 25 μl

0.05M phosphate buffer (pH 7.0) used as a carrier,

20 ng of the obtained telomeric DNA sequences and the indicated peptide in a molar ratio of peptides: DNK 100: 1 and higher.

Detection of the DNA-peptide complex was carried out by electrophoresis on a 1% agarose gel by staining with ethidium bromide and subsequent exposure to UV light. The delay (retardation) of migration of the DNA fragment in the gel indicated the formation of the complex.

The use of cosmetics can be carried out in various ways. The specified cosmetic product can be presented, for example, in the form of creams, ointments, gels, etc., intended for application to the skin, or solutions intended both for application to the skin (for example, using patches), and for subcutaneous administration useful for acting on dermal cells, for example fibroblasts. It is known that normal human somatic cells are able to divide only a limited number of times (the so-called

"Heiflik limit"). It has been proven that with each division of the somatic cell, as a result of the so-called “end-non-replication” problem, the length of telomeric DNA is reduced (Greider S. M., 1996, Appial Rev. Bioshet., 65, 337-365); when the telomere is shortened to a certain length, the cell ceases to divide, turning into a state senility, or senesensa, after which it ceases to function normally and dies.

When the proposed cosmetic product is introduced into the skin, telomeric DNA and the peptide associated with it penetrate into the cells due to the presence in the peptide of a component, such as PTD, which gives the complex containing DNA and peptide the ability to penetrate the cell membrane, or due to the liposome into which the DNA is introduced and a peptide and which is capable of transporting its contents across the cell membrane. The presence of a nuclear localization signal in the peptide allows the peptide-telomeric DNA complex to penetrate through the nuclear membrane into the cell nucleus. Penetrated into the nucleus of the cell, the telomere DNA sequence, using the enzyme systems present in the cell, attaches to the ends of the chromosomes, attaching them. As a result of lengthening of the telomeric regions of the chromosomes, the cell “rejuvenates”, i.e. becomes able to carry out more divisions.

Thus, the skin into which the cosmetic is introduced produces more young cells. As a result, the epidermal re-epithelialization process is accelerated, which provides the skin surface with younger and denser cells. In addition, due to the production of a large number of young cells, the epidermis is effectively maintained. Finally, the penetration of the telomeric DNA-peptide complex contained in the cosmetic product into the fibroblasts located in the dermis ensures the production of more young fibroblasts in it, which, unlike the old ones, produce more elastin fibers, which makes the skin more elastic. Providing the skin surface with young and densely spaced cells, maintaining the thickness of the epidermis and increasing skin elasticity provide an effective improvement in the functions and appearance of the skin. Compared to the frequently used transfer of DNA molecules by virus-based vectors, the transportation of the telomeric DNA sequence into a cell carried out using the proposed cosmetic product avoids undesirable ^' immunological reactions, toxic response and possible mutations.

Industrial applicability

This invention can be used in various skin care products used in cosmetology to improve the appearance of human skin.

Claims

Claim

1. A cosmetic product containing an active ingredient and a carrier, characterized in that the active ingredient contains a complex that includes a telomeric DNA sequence and its associated peptide having a nuclear localization signal, and which is able to penetrate through the plasma membrane of the cell.

2. The tool according to claim 1, characterized in that said peptide contains a protein transduction domain.

3. The tool according to claim 2, characterized in that said peptide contains an amino acid sequence

GRKKRRQRRRPPQRKKKRKV.

4. The tool according to claim 2, characterized in that said peptide contains the amino acid sequence RQIKIWFQNRRMKWKKRCKKKRKV.

5. The method according to claim 2, characterized in that said peptide contains the amino acid sequence GALFLGFLGААGSТМGАWSQРКSКRКV.

6. The tool according to claim 1, characterized in that said complex further includes a liposome into which said telomeric DNA sequence and said peptide are introduced.

7. A method of obtaining a cosmetic product, characterized in that

- synthesize the telomeric sequence of human DNA and - the resulting telomeric DNA sequence is combined with a peptide having a nuclear localization signal, with the formation of a complex capable of penetrating through the plasma membrane of the cell.

8. The method according to claim 7, characterized in that said peptide contains a protein transduction domain.

9. The method according to claim 7, characterized in that when the complex is formed, the telomeric DNA sequence and the peptide associated with it are introduced into the liposome.

10. The method according to claim 7, characterized in that a blood or tissue sample is taken from the person to whom the cosmetic product is to be applied, a total genomic DNA is obtained from it, and the specified telomeric sequence is synthesized as a matrix on its basis.